The cystine and methionine content of Montana range grass
by Earl W Turner
A THESIS Submitted to the Graduate Committee in partial fulfillment of the requirements for the
degree of Master of Science in Chemistry
Montana State University
© Copyright by Earl W Turner (1947)
Abstract:
The leaf proteins of several Montana range grasses are extracted using sodium dodecyl sulfate. The
advantages claimed for this method of extraction are simplicity, rapidity, and high yields of a final
product containing as high as 80% pure protein. Cystine and methionine are determined on the leaf
protein preparations using both chemical and micro biological assay methods. A comparison is made of
the results obtained by the various assay methods. Methionine and cystine values obtained by both
microbiological and chemical methods are found to be in good agreement and also consistant with
results obtained by other methods. Methionine and cystine are determined on Agropyron smithii and
Agropyron cristatum at two different stages of growth. The methionine and cystine contents of the
younger grasses are considerably higher than of the more mature plants. THE CYSTINE AND METHIONINE CONTENT OP
MONTANA RANGE GRASS
by
EARL W. TURNER
A THESIS
S u b m itte d t o th e G rad u ate Comm ittee
in
p a r t i a l f u l f i l l m e n t o f th e re q u ire m e n ts
f o r th e d e g re e o f
M aster o f S c ie n c e in C h em istry
at
M ontana S ta t e C o lle g e
C hairm an, E xam ining Comm ittee
Bozeman, Montana
May,
1947
//■3 7 f
2
TABLE OF COSTEHTS
Page
I.
II.
III.
IV .
' 'r
V.
T i.
y
ABSTRACT..........................
3
IKT ODUCT10» .
.
4
.
5
. .
HISTORICAL ® S W E
STATBVSST OF PROBLEM
21
EXFERIKEliTAL WOHK
22
P r o te I n E x t r a c t i o n . . . . . . . . . . . .
22
H y d ro ly s is o f P r o t e i n . . . . . . . . . . .
26
C o lo r im e tr ic D e te rm in a tio n o f C y s tin e . . .
26
C o lo r im e tr ic D e te rm in a tio n o f M eth io n in e •
34
M ic r o b io lo g ic a l D e te rm in a tio n o f C y s tin e •
56
M ic r o b io lo g ic a l D e te rm in a tio n o f M eth io n in e
45
D is c u s s io e o r e x p e t i ^ sk ta l a s s o u s
51
P r o te i n E x t r a c t i o n . . . . . . .
51
C y s tin e D e te rm in a tio n s . . . . .
66
S a th io n in e D e te r m in a tio n s . . • •
72
%
K
V II.
SUMAHT.....................
76
V III.
ACKMOWlEDGKEKT .
78
IX .
LITEEATUHS CITED
79
81286
- 3 •*
I.
ABSTRACT
The l e a f p r o te in * o f s e v e r a l MontarA ran g e g r a s s e s a r e e x t r a c t e d u sin g
sodium d o d e e y I s u l f a t e .
The a d v a n ta g e s claim ed f o r t h i s m ethod o f e x t r a c ­
t i o n a r e s i m p l i c i t y , r a p i d i t y , and h ig h y i e l d s o f a f i n a l p ro d u c t c o n ta in in g
a s h ig h a s 80% p u re p r o t e i n .
C y s tin e and m e th io n in e a re d e te rm in e d on th e
l e a f p r o t e i n p r e p a r a t io n s u sin g b o th c h e m ic a l and micro b i o l o g i c a l a s s a y
m e th o d s.
A co m p ariso n i s made o f th e r e s u l t s o b ta in e d by th e v a r io u s assay
m e th o d s.
M eth io n in e and c y s t i n e v a lu e s o b ta in e d b y b o th m ic r o b io lo g ic a l
and c h e m ic a l m ethods a r e found to be in good ag ree m en t and a ls o c o n s i s t a n t
w ith r e s u l t s o b ta in e d by o t h e r m e th o d s.
F e th io n liA and c y s tin e
a re d e te r­
mined on A gropyron s m it h ! ! and A gropyron c r i s t a t u m a t two d i f f e r e n t s ta g e s
o f g ro w th .
The m e th io n in e and c y s t i n e c o n te n ts of th e y o u n g e r g r a s s e s a r e
c o n s id e r a b ly h ig h e r th a n o f th e more m ature p l a n t s .
-
II.
4
-
ISTHODDCIIOS
The p re s e n c e o f th e s u l f u r c o n ta in in g am ino a c i d s , c y s tin e and m e th io n in e ,
i n p l a n t p r o t e i n s h as b e e n known f o r some t i m e .
S in c e th e s e amino a c id s a r e
q u i t e e a s i l y a l t e r e d by th e a c t i o n o f o r d in a r y h y d r o ly t ic r e a g e n ts and p ro b a b ly
by re a g e n ts em ployed a s p r o t e i n e x t r a c t a n t s , th e d e te r m in a tio n o f th e s e a d d s
i n p l a n t l e a f p r o t e i n s p r e s e n ts a s p e c i a l p ro b lem t o th e b i o l o g i c a l c h e m is t.
C y s tin e and m e th io n in e a r e e s s e n t i a l t o th e grow th o f many o rg a n is m s .
In v iew
o f th e s e f a c t s , a more e x a c tin g s tu d y o f m ethods o f d e te r m in in g th e s e amino
a c id s in p la n t p ro te in s is d e s ir e d .
I n th e p a s t , q u a n t i t a t i v e p ro c e d u re s f o r th e d e te r m in a tio n of m e th io n in e
and c y s t i n e have n o t b e e n s u f f i c i e n t l y a c c u r a te t o a f f o r d more th a n a p p r o x i­
m ate r e s u l t s .
A lth o u g h t h e r e a r e a number o f c h e m ic a l m ethods a v a i l a b l e f o r
th e e s t i m a t i o n o f m e th io n in e and c y s t i n e , many o f th e s e m ethods p r e s e n t
s e r i o u s d i f f i c u l t i e s w hich le a v e some d o u b t a s t o th e r e l i a b i l i t y o f th e
r e s u l t s o b ta in e d .
R e c e n tly much work h as b e e n done i n a n e f f o r t t o im prove
th e o l d e r m ethods and t o d e v e lo p new m ethods o f a n a ly s e s w h ich would g iv e
more c o n s i s t e n t and a c c u r a te r e s u l t s .
Of th e more r e c e n t l y d e v elo p ed m ethods
m i c r o b io l o g ic a l a s s a y in g o f f e r s i n t e r e s t i n g p o s s i b i l i t i e s .
In view o f th e s e d ev elo p m en ts and th e i n t e r e s t i n g p ro b lem s th a t a re
p r e s e n te d , i t was d e c id e d t o i n v e s t i g a t e th e d e s i r a b i l i t y o f th e m ic ro b io ­
l o g i c a l a s w e l l as th e c h e m ic a l m ethods o f a n a l y s i s .
The l e a f p r o t e i n
e x t r a c t s from tw o common v a r i e t i e s o f Montana, ran g e g r a s s , A gropyron s m ith ! !
and A gropyron o r l s t a t u m , w ere s e l e c t e d f o r t h i s s tu d y .
—6 —
III.
HISTORICAL RESUME
Tha L ise o v a ry o f ^ e th io n in a and C y s tin e i
Two s u l f u r d e r i v a t i v e s have b e e n found a s c o n s t i t u e n t s o f th e p r o t e i n
moI e e u I e .
These a r e c y s t i n e and M u e lle r 's t h i o a c id ( m e th io n in e ) .
C y stin e
was i s o l a t e d i n 1810 by W o lla s to n and was found t o be d i,J 3 -thio-*C -am ino
p r o p io n ic a c i d .
M B rner, O sborne and G u e s t, D ak in , O sboriw , H a r r is and o th e r s
found t h a t th e r e was a p r o b a b i l i t y o f s u l f u r compounds i n th e p r o t e i n m o le c u le
o t h e r th a n c y s t i n e .
I n 1922 M u e lle r i s o l a t e d from c a s e i n a new s u l f u r con­
t a i n i n g am ino a o i d , t o w h ich he gave th e e m p ir ic a l fo rm u la CnHggSHaO*.
Sub­
s e q u e n t w ork on t h i s am ino a c id by M u e lle r i n d i c a t e s t h a t t h e am ino a c id has
th e e m p e rio a l form ula CgHiiSKOg in s te a d o f Cu H22SN2O4 .
In 1928 B arg er and
Coyne showed t h a t M u e ll e r 's t h i o ao id had th e c o n s t i t u t i o n « a m in o -Y -m e th y l
t h i o l - n - b u t y r i c a c i d , CHg-S-CB2-CK2 -CH-COOH and gave i t th e name m e th io n in e .
NH2
P r o te i n E x t r a c ti o n Methods >
One o f th e m ost d i f f i c u l t problem s i n th e s tu d y o f l e a f - p r o t e i n s i s
th e e x t r a c t i o n and p u r i f i c a t i o n o f th e p r o t e i n w ith o u t s u b se q u e n t lo s s o f
am ino a o i d .
f o r m a tio n .
The lo s s o f amino a o id s o c c u rs m ost r e a d i ly when th e r e i s hum in
Kumin i s form ed when th e h y d r o ly s is i s c a r r i e d o u t i n tine
p re s e n c e o f c a r b o h y d r a te s .
To d e te rm in e th e p r o t e i n c y s ti n e c o n te n t i t i s n e c e s s a r y t o h y d ro ly s e
th e p r o t e i n w ith s tr o n g a c id o r w ith th e a p p r o p r ia te p r o t e o l y t i c enzym es.
As no method o f p r e p a r in g th e s e p r o t e i n s h a s h i t h e r t o been a v a i l a b l e , e a r l y
w o rk ers found i t n e c e s s a r y t o su b m it th e w hole g r a s s to a c id h y d r o ly s is o r
enzym ic d i g e s t i o n .
T h is m a n s i n e f f e c t t h a t th e p r o t e i n s i n th e l e a f
m a t e r i a l have b e e n t r e a t e d in th e p re s e n c e o f from 3-6 tim e s t h e i r own
~
6
«»
w e ig h t o f e e l l u l o s e , s u g a r s , o r g a n ic a c i d s , e t c . , and i t i s n o t a t a l l s u r ­
p r i s i n g t h a t u n d e r th o s e c o n d itio n s th e y w ere u n a b le t o a t t a i n s a t i s f a c t o r y
re s u lts .
H ow ever, by e x t r a c t i o n o f th e p r o t e i n s b e f o r e h y d r o ly s is f a i r l y
r e l i a b l e r e s u l t s may be o b ta in e d .
C h ib n a ll and Lugg have em ployed a method o f e x t r a c t i n g p r o t e i n s from
f r e s h l y o u t g r a s s w h ich in v o lv e s th e u se o f e t h e r - w a t e r a s th e p lasm o ly s in g
a g e n t and a p r e s s f o r s q u e e z in g o u t th e s o lu b le p ro to p la s m ic p r o t e i n s .
r e s u l t a n t f l u i d i s t r e a t e d w ith a c id t o f l o c c u l a t e th e p r o t e i n s .
The
The p r e c ip ­
i t a t e d p r o t e i n s w ere p u r i f i e d and s u b je c te d t o a n a l y s i s f o r th e v a r io u s am ino
a c id s .
The y i e l d o b ta in e d com pared w ith th e ^ o t a l p r o t e i n i n th e l e a f was
v e r y low y a t th e a s s u m p tio n was r.ade t h a t th e p r o te i n s w h ich rem ained une x t r a c t e d i n th e l e a f t i s s u e w ere o f th e same c o m p o s itio n a s th o s e which had
b een e x t r a c t e d .
Lugg found o n ly s l i g h t v a r i a t i o n s In th e amino a c id com posi­
t i o n b etw ee n th e v a r io u s g r a s s e s he ex am in ed .
A n o th er method f o r th e e x t r a c t i o n o f l e a f p r o t e i n s i s th e method o f
S s iith ( 3 9 ) .
S m ith has u sed sodium d o d e e y l s u l f a t e , d i g i t o n i n , and b i l e s a l t s
a s th e d i s p e r s i n g a g e n t .
P r e lim in a r y e x p e rim e n ts w ith so d iu m d o d e e y l s u l f a t e
have b een em ployed a t M ontana S t a t e C o lle g e A g r i c u l t u r a l E x p erim en t S t a t i o n
and i t has b e e n found t h a t much h ig h e r y i e l d s o f e x t r a c t e d p r o te in s can be
o b ta in e d th a n i s p o s s ib le by th e C k ib n a ll-L u g g m eth o d .
F u rth e rm o re , th e
method seem s a p p l ic a b l e t o d r ie d g r a s s e s .
A t h i r d m ethod f o r th e e x t r a c t i o n o f g r a s s p r o te i n s h a s been s u g g e s te d
b y A lb an ese ( I ) and Mazur and C la rk e ( 2 9 ) .
s o l u t i o n i s used a s th e e x t r a c t a n t .
I n t h i s method 90% fo rm ic a c id
Mazur and C la rk e have em ployed t h i s
e x t r a c t a n t i n t h e i r s t u d i e s on th e p r o t e i n c o m p o s itio n o f m arin e a l g a e .
-
7
-
Albemese h as e x t r a c t e d l e a f p r o t e i n s from su c h p la n ts a s s p in a c h , c a b b a g e ,
and c e l e r y by t h i s means and h as b e e n a b le t o o b ta in a y ie ld
th e t o t a l p r o t e i n .
o f 90-96/5 o f
The e x t r a c t e d p r o t e i n i s rem a rk ab ly f r e e o f c a rb o h y d ra te
c o n ta m in a tio n w h ich i s a d e c id e d a d v a n ta g e when th e p r o t e i n p r e p a r a t i o n i s
t o b e a n a ly z e d f o r i t s c o n s t i t u e n t am ino a c i d s .
H y d ro ly s is o f th e P r o te i n l a t e r i a l i
h y d r o ly s is by a c i d s , a l k a l i e s o r p r o t e o l y t i c enzymes h a s p ro v ed t o be
th e b e s t means o f I n v e s t i g a t i n g th e c o m p o s itio n o f th e p r o t e i n m o le c u le .
1820 B raoonnot h y d ro ly z e d g e l a t i n by b o i l i n g w ith d i l u t e s u l f u r i c a c i d .
In
Be­
tw een 1850 and 1875 g i t t h a u s e n , H la s iw e ti and Heiberaann a s w e ll a s l a t e r i n ­
v e s t i g a t o r s have f a v o re d h y d r o c h lo r ic a c id a s a h y d r o ly z in g a g e n t .
The
a c t i o n o f v e g e ta b le enzymes on p r o t e i n s h as b e e n s tu d ie d e s p e c i a l l y by S ch u lz e
and h i s c o -w o r k e r s , w h ile a b o u t 1900 th e a c t i o n o f an im al enzymes was e x te n ­
s i v e l y s tu d ie d by Ktihne, K o sseI , K u teo h er and o t h e r s .
In 1912, Van S ly k e fou n d t h a t co m p lete h y d r o ly s is was p roduced by h e a t ­
in g th e p r o t e i n w ith 3 H h y d r o c h lo r ic a c id in a n a u t o c l a v e .
The fo rm er
i n v e s t i g a t o r s do n o t s t a t e th e q u a n t i t y o f a c id used b u t Van S ly k e used t h i r t y t h r e e tim e s more a c id th a n p r o t e i n .
Van SlykB showed f u r t h e r t h a t co m p lete
h y d r o ly s is was e f f e c t e d e i t h e r oy h e a t in g a t 100° f o r 48 h o u rs w ith 20% a c id
o r a t 160° w ith S N a c i d .
H en riq u es and G ja ld b ik (1910) found t h a t th e
h y d r o ly s is o f eg g alb u m in was n o t co m p lete a f t e r h e a t in g f o r 12 h o u rs w ith
c o n c e n tr a te d a c i d , b u t was co m p lete on h e a t in g a t IbO0 f o r 1 .5 h o u rs in an
a u to c la v e w ith 3 8 a c i d .
They found no a d v a n ta g e i n h e a t in g a t 160° f o r a
lo n g e r ti m e ; a t ISO0 In a n a u to c la v e d e c o m p o sitio n o f th e amino a c id s o c c u rre d
- 8 —
w i t h t h e f o r m a t io n o f auunonia.
Z e l i n s k y and S sa d ik o w ( 1 9 2 5 ) w ere a b le t o
sh ow t h a t c o m p le t e h y d r o ly s i s may b e e f f e c t e d w it h v e r y w e a k a c i d s .
sta te
Thqy
t h a t p r o t e i n s may be c o m p l e t e l y h y d r o ly s e d b y h e a t i n g i n an a u t o c l a v e
a t 1 8 0 ° f o r t h r e e t o s i x h o u r s w i t h 4 , 3 , 2 , I o r e v e n 0 .5 < h y d r o c h lo r i c
a c id .
I t h as b e e n fo u n d t h a t t h e am ount o f c y s t i n e r e c o v e r e d from h yd ro­
l y s a t e s d e p e n d s som ew hat upon th e a c i d u sed f o r h y d r o l y s i s .
Ahen m ix tu r e s
o f f e m i e and h y d r o c h lo r i c a c i d s w ere u se d t o h y d r o ly s e i n s u l i n some i n v e s t i ­
g a t o r s fo u n d h a l f a g a i n a s much c y s t i n e a s w hen h y d r o c h lo r i c a c i d was u sed
a lo n e .
B a e r n s t e in
( 6 ) h a s a d v o c a te d th e u s e o f HI b u t t h i s
i n f e r i o r t o t h e fo r m ic a c id m ix tu r e by du V ig n e a u d .
ta in ly not as s e n s itiv e
S u lliv a n
regarded as
t o s t p r o te in s are c e r ­
to h y d r o ly s is c o n d it io n s as i s
in s u lin .
Hess and
(1 8 ) h a v e i n v e s t i g a t e d t h e r a t e o f l i b e r a t i o n o f c y s t i n e from
p r o t e i n s by a c i d h y c r o l y s l s .
S i x p r o t e i n s v a r y in g c o n s i d e r a b l y in t h e i r
c y s t i n e c o n t e n t w e re c h o s e n f o r t h e e x p e r im e n t .
The p r o t e i n s w e re h y d ro ­
ly z e d w i t h 20% HCI , HGI-KCOOH m i x t u r e s , and w i t h H I.
m ethod o f h y d r o l y s i s
R e g a r d le s s o f t h e
u s e d , th e maximum, v a l u e s o f a l l t h r e e m eth od s w ere
fo u n d t o be p r a c t i c a l l y
s u lte d .
is
I d e n tic a l.
The HI h y d r o l y s i s
W ith KI h y d r o l y s i s no hum in fo r m a tio n r e ­
i s by f a r th e more r a p id p r o c e d u r e and g i v e s m axi­
mum v a l u e s i n t h e s h o r t e s t p o s s i b l e t i r e s .
I t has th e d i s a d v a n t a g e s , b o s e v e r ,
t h a t th e Hgi'Og and HI m ust be d r i v e n o f f b e f o r e t e s t i n g . f o r c y s t i n e and th e
h y d r o ly s a t e c o n t a i n s o n ly c y s t e i n e w h ic h p r e v e n t s th e s e p a r a t e e s t im a t i o n
o f c y s t i n e and c y s t e i n e .
The HCl h y d r o l y s i s h a s th e a d v a n ta g e t h a t i t i s
p o s s i b l e t o s tu d y b o th c y s t i n e an c c y s t e i n e
m ate m e th io n in e c o l o r i n e t r i o a l l y .
i n th e h y d r o ly s a t e and t o e s t i ­
W ith HCl and th e HCl-KCCXSi m ix tu r e some
hum in was form ed b u t t h e r e w as n o e v id e n c e o f l o s s o f c y s t i n e ,
h y d r o ly s is
-
w ith 2 0
9
-
HCl w as fou n d t o be more r a p id th a n w ith t h e HC1-BC0GH m ix tu re #
S u l l i v a n ' s c o l o r i m e t r i c m ethod was u sed f o r th e e s t i m a t i o n o f c y s t i n e .
R e c e n t ly many i n v e s t i g a t o r s h a v e fou n d t h a t a u t o c l a v i n g w i t h d i l u t e
h y d r o c h lo r i c a c i d ga v e v e r y s a t i s f a c t o r y
S e h w e ig e r t ( 3 7 ) and S t o k e s
r e s u lts .
B a r t o n - S r ig h t ( 8 ) a l s o
( 4 2 ) f a v o r t h i s m ethod o f h y d r o l y s i s .
B a r to n -
',Yright h a s u se d th e f o l l o w i n g p r o c e d u r e f o r t h e p r e p a r a t io n o f p r o t e i n h y d r o ­
l y s a t e s f o r m e th io n in e and c y s t i n e a s s a y s *
A one gram sa m p le o f f i n e l y
g rou n d m a t e r i a l i s h y d r o ly s e d b y a u t o c l a v i n g f o r 5 - 6 h o u r s a t 16 pounds p r e s ­
s u r e w i t h 2 6 m l. o f 2 . 6 S HC1.
l e s s th a n f i v e h o u r s ,
a c e ta te s o lu tio n i s
He s t a t e s t h a t h y d r o l y s i s i s
th e h y d r o ly s a t e
in c o m p le te In
i s c o o l e d , 2 m l. o f 2 ^
16 so d iu m
added and t h e pH a d j u s t e d t o 4 . 6 w i t h so d iu m h y d r o x id e
and t h e s o l u t i o n made up t o v o lu m e .
M ethods f o r C y s t in e D e t e r m in a t io n
S u l l i v a n * s L eth od f o r t h e D e te r m in a tio n o f C y s t in e *
The l i t e r a t u r e
on S u l l i v a n ' s
r e a c t i o n f o r c y s t i n e h a s a t t a i n e d f o r m i­
d a b le p r o p o r t io n s and a num ber o f m o d i f i c a t i o n s h ave b e e n p r o p o s e d , c o n s i ­
d e r a b le a c c u r a c y i s
c la im e d f o r m ost o f th em by t h e i r a u t h o r s , th o u g h r e ­
g a rd ed a s in a d e q u a te b y o t h e r s .
The r e a c t i o n w h ic h i s h i g h l y s p e c i f i c
fo r
c y s t e i n e g i v e s a red c o l o r w it h I , 2 n a p h t h o q u i n o n e - 4 - s u l f o n a t e i n a n a l k a ­
lin e
r e d u c in g m edium .
S u l l i v a n ' s p r o c e d u r e f o r th e e s t i m a t i o n
o f c y s tin e
c o n s i s t s e s s e n t i a l l y i n r e d u c in g t h e c y s t i n e t o c y s t e i n e and a l l o w i n g th e
c y s t e i n e t o r e a c t w it h th e c o l o r r e a g e n t i n a n a l k a l i n e s o l u t i o n .
S u l l i v a n a d v o c a t e s th e a d d i t i o n o f a s m a l l amount o f c y a n id e b e f o r e t h e
re a g en t t o s t a b i l i s e th e c o lo r .
The p r o c e d u r e f o r c y s t i n e c o n s i s t s
In f i r s t
t r e a t i n g t h e s o l u t i o n w it h s u f f i c i e n t c y a n id e t h e n p r o c e e d in g a s w i t h c y s t e i n e .
10
-
P r u n ty 1S m o d if ic a tio n o f S u l l i v a n 's m ethod has been u sed by C h ib n a ll
(5 6 ) f o r th e d e te r m in a tio n o f c y s t i n e and c y s te i n e i n l e a f p r o te i n s *
P ru n ty *8 m o d i f ic a t io n i s much th e san e a s th e o r i g i n a l S u lli v a n method
e x c e p t f o r th e a d d i t i o n o f a few mg. o f s in e d u s t and a d ro p o f c o n c e n tr a ­
te d h y d r o c h lo r ic a c i d w h ich I s u sed t o h e lp m a in ta in a re d u c in g en v iro n m en t
i n th e s o l u t i o n .
E x p e rie n c e s w ith S u l l i v a n 's m ethod w ere found by Lugg (2 7 ) t o be un­
s a t i s f a c t o r y i n th e f o llo w in g r e s p e c ts *
( I ) th e d e p th o f c o l o r i s v a r i a b l e
and f a r from p r o p o r t i o n a l t o th e am ount o f c y s t e i n e and c y s t i n e p re s e n t*
(2 ) ev en when com pared w ith a s ta n d a r d o f t h e same c o l o r r e s u l t s a r e o f te n
ir r e g u la r *
(5 ) th e p re s e n c e o f r e l a t i v e l y c o n s id e r a b le am ounts o f o th e r
am ino a c id s d im in is h e s t h e amount o f re d c o l o r due t o c y s te i n e and y e llo w
i n t e r f e r e s when a p u re c y s t i n e o r c y s t e i n e i s used f o r a s t a n d a r d .
Lugg
s u g g e s te d two m ethods w hich m ig h t im prove th e a c c u ra c y o f th e method* ( I )
e
m a in ta in th e same pH i n th e unknown a s i n th e s ta n d a rd * (2 ) add some am ino
a c id i n c o n s id e r a b le e x c e s s t o b o th th e unknown and t o th e s t a n d a r d .
It
was found p o s s i b le t o e f f e c t b o th o b je c tiv e s by a d d in g g ly c in e w ith ap p ro ­
p r i a t e amou nts o f a l k a l i .
O th er m ethods w h ich have b een used f o r th e e s t i m a t i o n o f c y s ti n e i n ­
c lu d e Okuda and B a e r n e te i n ' s lo d o m e trie d e t e r m in a tio n s .
c y c t in e t o o y s t e i c a c i d by b r ornate i n a c id s o l u t i o n .
t e r f e r e w ith t h i s method o f d e t e r m in a tio n ,
Okuda o x id is e d
O th e r am ino a c id s i n ­
l a t e r he t i t r a t e d th e c h a r c o a l
d e c o l o r is e d h y d r o ly s a te w ith KIOg i n a c id s o l u t i o n c o n t a in in g K I, o x id i s i n g
c y s te in e to c y s tin e .
B a e r n s te in (2 ) t o a v o id lo s s e s i n c h a r c o a l added e x ­
c e s s io d in e and u sed g a s o m s tr ie m ethods t o d e te rm in e th e e x c e s s io d i n e .
11
-
T h i s , h o w ev er, le d to h ig h v a lu e s f o r c y s t e i n e .
B a e r c e te ln (6 ) recosucende
th e Okuda m ethod on HI h y d r o ly s a t e s th o u g h o n ly c y s te i n e and c y s ti n e co u ld
be e s tim a te d i n t h i s w ay, b u t humin i s a b s e n t and th e r e i s no n e c e s s i t y t o
u se c h a r c o a l o r o th e r d e c o l o r i z i n g a g e n t s .
The F o lin m ethod f o r th e d e te r m in a tio n o f c y s ti n e h as a l s o been w ide­
ly u s e d .
Lugg1S m o d i f ic a t io n o f th e F o lin - S a r e n z i method ( 2 6 ^ i n p r i n c i p l e
u t i l i z e s th e r e d u c t io n o f molybdenum f r e e p h o s p h o - lf l- tu n g s tic by c y s te i n e
and in s u r e s a r i g i d c o n t r o l o f pH by th e u s e o f a c i t r a t e b u f f e r a t a pH
o f 6 .7 .
T h is a v o id s th e d is a d v a n ta g e s i n th e c o lo r p r o d u c tio n o f a n a l k a ­
lin e r e a c tio n .
In th e o r i g i n a l F o lin -M a re n z i m ethod, p r e c i p i t a t i o n o f te n
o c c u rre d d u r in g th e d e te r m in a tio n and t h e r e w ere no means o f c o r r e c t i n g
f o r e x tra n e o u s re d u c in g r e a g e n t s .
In Lugg’ s m o d if ic a tio n p r e c i p i t a t i o n i s
a v o id e d and an in g e n io u s c o r r e c t i o n i s a p p lie d f o r re d u c in g r e a g e n ts o th e r
th a n -SH- g ro u p s .
In th e p re s e n c e o f HgClg c y s te in e p ro d u c e s no c o lo r
w ith p h o s p h o tu n g s tic a c id and th e c o l o r d ev e lo p e d by o t h e r r e d u c e rs i s
m e a su re d .
b ility .
T h is method i s r a p id and c a p a b le o f a h ig h d e g re e o f r e p r o d u c i­
In U iggt S m o d i f ic a t io n o f S u l l i v a n ’ s method g r e a t c a re m ust be
e x e r c is e d t o izisu re t h a t th e s o l u t i o n s , a f t e r p o u rin g i n t o th e c o lo r im e te r
c u p s , a r e c o m p le te ly red u ced b e f o r e c o m p a ris o n .
B a ile y (7 ) h as u sed v a r io u s m ethods f o r t h e d e te r m in a tio n o f o y s tin e
i n p r o t e i n s and h as a d v o c a te d Luggt s m o d if ic a tio n o f th e F o lin -M a re n z i
(2 6 ) a s a m easure o f th e t o t a l d i s u l f i d e and f o r t h e e s t i m a t i o n o f c y s t i n e .
He s t a t e s t h a t Luggt S m o d if ic a tio n o f th e F o lin -M a re n z i m ethod i s ra p id
and c a p a b le o f a h ig h d e g re e o f r e p r o d u c i b i l i t y and t h a t th e c o l o r m a tch in g
i s more p r e c i s e th a n S u l l i v a n 's m ethod o r i t s m o d i f i c a t i o n s .
I n view o f
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12
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t h i s In fo rm a tio n and th e fo r e g o in g c o n s id e r a tio n s t h i s method would a p p e a r
t o be th e m ost d e s i r a b l e from th e s ta n d p o in t o f a c c u ra c y and r e p r o d u c i b i l i t y .
The f a c t o r s r e s p o n s ib le f o r e r r o r s i n th e d e te r m in a tio n o f c y s t i n e may
now c o n v e n ie n tly be sum m arized t
( I ) e r r o r s i n th e method a d o p te d , e . g . , i n ­
h i b i t i o n o f c o l o r by e x tra n e o u s r e d u c e rs a s i n th e S u l l i v a n m ethod; (2 )
l o s s e s d u r in g h y d r o l y s i s .
K e th lo n in e D e te rm in a tio n s *
B a e r n s te i n (3 ) (4 ) has shown t h a t on h e a tin g w ith BI m e th io n in e i s d e ­
ne th y la te d and may be e s tim a te d a s th e v o l a t i l e io d i d e .
In a l a t e r p a p e r
(6 ) he h as shown t h a t m e th io n in e may be e s tim a te d a f t e r i t has b een con­
v e r te d t o th e th i o l a o t o n e o f h o m o cy stein e by b o i l i n g w ith H I.
B a e r n s te in o b ta in s p r a c t i c a l l y q u a n t i t a t i v e r e s u l t s w ith t h i s m ethod.
K a s s e ll and Brand (2 2 ) have n o t found co m p lete r e c o v e r ie s and th e h y d ro been
l y s i s w ith HI h a v e / c r i t i c i z e d on th e g ro u n d s t h a t i t c a u s e s g r e a t e r e v o lu ­
t i o n o f v o l a t i l e io d id e and HgS from i n s u l i n (w hich c o n ta in s no m e th io n in e )
th a n d o es BCl h y d r o l y s i s .
M cCarthy and S u l l i v a n (SO) p ro p o sed a method f o r th e d e te r m in a tio n o f
m e th io n in e b ased on th e f o r m a tio n o f red c o l o r form ed when a l k a l i n e s o l u ­
t i o n s o f m e th io n in e and sodium n i t r o p r u s s i d e a r e a c i d i f i e d .
They n o te d ,
h o w ev er, t h a t b o th try p h to p h a n e and h i s t i d i n e produce a red c o l o r u n d er
s im ila r c o n d itio n s .
They d e s tr o y e d th e try p to p h a n e w ith a c id h y d r o ly s is
and s t a t e d t h a t th e a d d i t i o n o f g ly c in e would e lim in a te th e c o l o r produced
by h i s t i d i n e .
Csonka and D enton (1 0 ) have d e te rm in e d th e m e th io n in e c o n te n t o f
p r o t e i n s and fo o d s u s in g th e M c C a rth y -S u lliv a n c o l o r i m e t r i c m ethod.
They
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15
—
u sed a Coleman s p e c tr o p h o to m e te r t o e s t a b l i s h s p e c t r a l tr a n s m is s i o n c u rv e s
f o r th e red c o l o r form ed*
Csonke and D enton found t h a t th e s to i c h i o m e t r i c
r e l a t i o n s h i p o f 2 s i was n o t v a l id when g ly c in e was added t o th e r e a c tio n
m ix tu re *
They fou n d t h a t g ly c in e c o n s id e r a b ly in c r e a s e d t h e tr a n s m is s io n *
T h is was f i r s t th o u g h t t o be cau sed by a r e a c t i o n betw een g ly c in e and sodium
n i t r o p r u s s i d e , ho w ev er, f u r t h e r i n v e s t i g a t i o n r e v e a le d t h a t th e g ly o in e
a l s o d e c re a s e d th e c o lo r p ro d u ced by th e m e th io n in e n l t r o p r u s s i d e r e a c tio n *
Tlw common io n e f f e c t i s a p la u s a b Ie e x p l a n a ti o n and s l i g h t l y in c re a s e d
am ounts o f sodium n l t r o p r u s s i d e i s a p p a r e n tly an a d v an tag e*
Tlw b e s t a p ­
p ro a c h t o th e p roblem w ould be t o s e p a r a t e m e th io n in e from th e o th e r
m a t e r i a l s in th e p r o t e i n h y d r o ly s a te s in c e h i s t i d i n e and o t h e r amino a c id s
e f f e c t th e r e a c t i o n m i x tu r e .
Kolb and T o e n c ie s (2 3 ) w ere a b le t o p r e c i p i t a t e m e th io n in e q u a n t i t a t i v e ­
ly w ith m ercu ry s a l t s from a s o l u t i o n c o n t a in in g no o th e r am ino a c i d s .
When t h i s was done w ith a p r o t e i n h y d r o ly s a te o th e r amino a c id s w ere a ls o
p re c ip ita te d ,
ta g e .
th u s th e p r e c i p i t a t i o n w i t h m ercury s a l t s i s o f no advan­
H i s t i d i n e may be p r e c i p i t a t e d w ith p h o e p h o tu n g stio a c i d and i t i s
th e n p o s s i b le t o use th e n i t r o p r u s s i d e t e s t f o r m e th io n in e w ith o u t th e
a d d itio n o f g ly c in e .
Grau one A lm q u ist (1 6 ) used th e IsC C a rth y -S u lliv a n c o l o r i m e t r i c method
f o r t h e e s t i m a t i o n o f m e th io n in e in f e e d s t u f f * .
They used b o th a c id and
e n zy m atic m ethods o f h y d r o ly s is and w ere u n a b le t o o b ta in co m p lete h y d ro ­
l y s i s o f th e p r o t e i n m a t e r i a l , th u s found i t n e c e s s a ry t o assum e t h a t th e
s o lu b le p r o t e i n was r e p r e s e n t a t i v e o f th e t o t a l p r o t e i n .
D e s p ite in ­
co m p lete h y d r o ly s is th e y w ere a b le t o o b ta in c o n s i s t e n t r e s u l t s u s in g th e
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14
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M c C a rth y -S u lliv a n m eth o d «
M ic r o b io lo g ic a l A ssaya i
The a b i l i t y o f c e r t a i n B io ro o rg an iam e t o grow on a s y n t h e t i c medium
p e rm its th e d ev elo p m en t o f a q u a n t i t a t i v e d e te r m in a tio n f o r e a c h o f th e
e s s e n t i a l c o n s t i t u e n t s o f th e medium,
ffith th e use o f su ch a c h e m ic a lly
d e f in e d medium t h i s method o f a s s a y h as b een w id e ly used th e l a s t few
y e a r s f o r th e q u a n t i t a t i v e d e te r m in a tio n o f v i t a m i n s .
R ecen t r e p o r ts i n
th e l i t e r a t u r e i n d i c a t e t h a t m i c r o b io l o g ic a l a s s a y in g may be a much
s im p le r and more a c c u r a te way t o d e te rm in e am ino a c id s th a n many o f th e
c h e m ic a l m ethods i n u se a t th e p r e s e n t ti m e .
One o f th e f i r s t m ic r o b io l o g ic a l a s s a y m ethods t o a c h ie v e re a s o n a b le
s p e c i f i c i t y was d e v is e d by 5 o h o p fe r i n 1955 f o r v ita m in B1 u s in g th e mold
F h y o o m y o ea-G lak eslean u s.
D e s p ite c o n tin u e d s tu d y and m o d if ic a tio n h i s
method has n o t b e e n w id e ly u s e d .
S n e l l and S tro n g (1 8 3 9 ) d ev elo p ed a m ic r o b io lo g ic a l m ethod f o r th e
d e t e r m in a tio n o f r i b o f l a v i n w hich was r a p id l y a c c e p te d and so o n g ain ed
w id e s p re a d u s e .
T h is was th e f i r s t method i n w h ich l a c t i c a c id p ro d u c in g
b a c t e r i a w ere u s e d .
As know ledge c o n c e rn in g th e g ro w th re q u ire m e n ts o f
l a e t i e a c id b a c t e r i a in c r e a s e d th e y w ere u t i l i s e d f o r th e a s s a y o f a d d i­
t i o n a l v it a m i n s .
W ith th e know ledge g a in e d c o n c e rn in g th e v ita m in r e q u i r e ­
m ents o f l a c t i c a c id b a c t e r i a i t became p o s s i b le to s tu d y th e n it r o g e n r e ­
q u ire m e n ts o f th e g r o u p .
F u r th e r i n v e s t i g a t i o n r e v e a le d t h a t a v a r i e t y o f
am ino a c id s w ere n e c e s s a r y f o r g ro w th .
The s p e c i f i c i t y o f th e d i f f e r e n t
am ino a c i d s was s tu d ie d and f u r t h e r i n v e s t i g a t i o n showed t h a t t h i s r e q u i r e ­
m e n t, in many i n s t a n c e s , c o u ld be a a d e th e b a s i s f o r q u a n t i t a t i v e m ethods
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15
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f o r th e d e t e r m in a tio n o f th e s e am ino a c id s *
N u tritiv e
H eq u ireE en ts o f K ie ro o rg a n ie m s t
The i d e a l medium f o r m i c r o b io l o g ic a l a s s a y sh o u ld c o n t a i n a l l sub­
s ta n c e s n e c e s s a r y o r s t i m u l a t o r y t o th e g ro w th o f th e m icro o rg an ism s b e in g
u s e d , b u t be c o m p le te ly f r e e o f th e n u t r i l i t e t o be d e te r m in e d .
T h is
i d e a l s i t u a t i o n i s a p p ro ac h ed more o r l e s s c l o s e l y by a s s a y m ethods no# in
u se*
An in tim a te know ledge o f th e grow th re q u ire m e n ts and optimum c o n d i­
t i o n s f o r g ro w th o f th e t e s t o rg an ism i s n e c e s s a r y b e f o r e a s a t i s f a c t o r y
q u a n t i t a t i v e a s s a y can be d e v e lo p e d .
The g e n e r a l n u t r i t i o n a l re q u ire m e n ts o f l a c t i c a c id b a c t e r i a may be
sum m arised a s f o llo w s t
( I ) e n e rg y s o u rc e - c a rb o h y d ra te ( g lu c o s e ) j (2 )
n i t r o g e n s o u rc e - am ino a c i d s i (3 ) a c c e s s a r y s u b s ta n c e s - v ita m in s and
o t t e r g ro w th f a c t o r s ; (4 ) m in e r a l s a l t s ; (5 ) b u f f e r .
F o r t h e i r en erg y
s u p p ly th e l a c t i c a c id b a c t e r i a depend upon th e p re s e n c e o f a fe rm e n ta b le
c a r b o h y d r a te .
Homo-f e r o e n t iv e o rg an ism s su c h a s L a c to b a c illu s a r a b i n o s u s ,
L a c to b a c i llu s d e l b r u c k l i and S tr e p to c o c c u s faeces.H s a r e a b le t o c o n v e r t
g lu c o s e a lm o s t q u a n t i t a t i v e l y i n t o l a c t i c a c i d .
G lucose
----------- ► 2 l a c t i c a c id
R e te ro fe rm e n tiv e o rg an ism s su c h a s L eueonostoo c te n s e n te ro id c s and L ae to b a c i l l u s f e n r e n t l p roduce o th e r p ro d u c ts from g lu c o se a s w e l l a s l a c t i c
a c i d , such a s e t h a n o l , COg e t c .
The optimum pK f o r most o f th e s e o rg a n ­
ism s i s b etw ee n 6 -7 a lth o u g h grow th w i l l c o n tin u e u n t i l a pH o f ab o u t 4 i s
re a c h e d .
To p r e v e n t th e l a c t i c a c id form ed from i n h i b i t i n g g row th a s u i t ­
a b le b u f f e r m ust be added t o th e medium .
A c e ta te was found e s p e c i a l l y
s u i t a b l e f o r t h i s p u rp o se by S n e l l , S tro n g and P e te rs o n and h as been w id e ly
-
u sed .
16
No e x t e n s i v e s t u d y o f th e m in e r a l r e q u ir e m e n ts o f l a c t i c a c i d b a c ­
t e r i a h a s b een m a d e.
For some b a c t e r i a ^ v a r i a t i o n s
h a v e b e e n fou n d t o im p rove g r o w t h .
i n t h e m in e r a l s a l t s
L uckey ( 2 6 ) sh o w e d , f o r e x a m p le , t h a t
t h e a d d i t i o n o f p o ta s s iu m s a l t s g r e a t l y im p roved th e g r o w th o f S t r e p t o c o c ­
cu s f a e c a lis .
p rove h e l p f u l .
K o d i f i o a t i o n o f th e m in e r a ls u se d i n o t h e r m ed ia m ig h t
One i s a b l e t o o b t a i n w h at i s
known a s a s ta n d a r d g ro w th
c u r v e by a d d in g t o th e s y n t h e t i c medium d i f f e r e n t i a l am ou n ts o f th e
n u t r i I i t e t o b e d e t e r m in e d .
The m e d ia t u b e s c o n t a i n i n g v a r y i n g am ounts
o f th e a m in e a c i d b e i n g t r e a t e d a r e t h e n i n o c u l a t e d w i t h t h e t e s t o r g a n is m ,
i n c u b a t e d , and th e l a c t i c a c id p ro d u ced i s t h e n t i t r a t e d w i t h a sta n d a r d
b a s e e . g . , 0 . 1 R S a OH.
The s ta n d a r d g ro w th 4urv© may t h e n be o b ta in e d
by p l o t t i n g th e e c . o f b a s e v s . m ic r o g r a s s o f am no a c i d p e r t u b e .
a s s a y o f p r o t e i n h y d r o l y s a t e s th e am ino a c i d b e i n g t r e a t e d
fo r is
F or th e
l e f t out
o f th e s y n t h e t i c medium and r e p la c e d b y v a r y in g c o n c e n t r a t i o n s o f th e p ro ­
t e i n h y d r o ly s a t e .
S i n c e t h e s e o r g a n ism s a r e a b le t o u t i l i z e
s u c h am in e
a c i d s i n th e form o f t h e i r p e p t i d e s , t h e am ino a c i d s may b e d e te r m in e d o v e n
i n i n c o m p l e t e l y h y d r o ly z e d p r o t e i n s .
and one i s
The l a c t i c a c i d
is
t it r a t e d as b e fo r e
r e a d i l y a b le t o c a l c u l a t e t h e am ount o f th e am ino a c i d p r e s e n t
i n th e h y d r o l y s a t e .
I t i s n e c e s s a r y t o e x p e r im e n t w i t h s y n t h e t i c m ed ia o f
v a r i o u s c o m p o s it io n s i n o r d e r t o o b t a i n a " p e r f e c t medium* i n w h ich th e
o rg a n ism w i l l a t t a i n maximum g ro w th u n d er optim um c o n d i t i o n s .
I any r e f e r ­
e n c e s a r e g i v e n i n t h e l i t e r a t u r e o f m e d ia w h ich h ave b e e n fo u n d s a t i s f a c ­
t o r y f o r t h e s e o r g a n is m s .
A lth o u g h m e d ia g i v e n f o r th e same o r g a n ism may
c o n t a i n t h e same b a s i c c o m p o n e n ts , th e p e r c e n t a g e c o m p o s i t i o n may v a r y c o n ­
s i d e r a b l y a c c o r d in g t o d i f f e r e n t r e f e r e n c e s .
T h is v a r ia n c e i n c o m p o s it io n
ie
l a r g e I y due t o th e d i f f e r e n c e
fe r e n t w ork ers.
The d i f f i c u l t y
1 7 -
In p u r i t y o f t h e am ino a c i d s u sed b y d i f ­
o f p r e p a r in g a s t a n d a r d is e d r e d lu m may be
m in im is e d b y t h e u s e o f p u re am ino a c i d s and v itim in s w h ic h a r e now a v a i l a b l e .
L a o to c a c illu s a r a c in o s u a :
P o l l a c k and L in d e r ( 5 5 ) h ave show n t h a t a s y n t h e t i c medium, com posed o f
tw e n t y am ino a c i d s , s a l t s , g l u c o s e , so d iu m a c e t a t e , v i t a m i n s and o th e r
g r o w th f a c t o r s w i l l s u p p o r t th e g ro w th o f L a c t o b a c i l l u s a r a b i n o s u s .
S n e ll
( 4 0 ) h a s show n t h a t a t l e a s t tw o s t r a i n s o f t h e o r i g i n a l c u l t u r e o f l r a r a b in o s u s e x i s t ;
one n e e d s p a r a - a m ic o b e n s o ie a c i d , t h e
o t h e r w h ic h i s a m u ta n t
does n o t.
Shankman ( 3 3 ) h a s s t u d i e d th e am ino a c i d r e q u ir e m e n ts o f L -a r a b ln o su a
and fo u n d t h e f o l l o w i n g am in o a c i d s t o be e s s e n t i a l n u t r i l l t e e t
• C y s t in e
T ry p to p h a n e
I s o le u o in e
g lu t a m ic a c id
• M e th io n in e
L e u c in e
V a lin e
T h r e o n in e
The l a c t i c a c i d p ro d u ced b y th e f e r m e n t a t i o n w as t i t r a t e d w i t h 0 , 1 N NhOH
and th e t i t r a t i o n w as found t o b e r e p r o d u c a b le w i t h i n 0 . 1 m l . u s in g b r o o
th y m o l b lu e f o r t h e i n d i c a t o r .
B l e r ( 1 9 ) h a s made m i c r o b i o l o g i c a l a n a l y s i s
o f a n im a l p r o t e i n s em p lo y ­
i n g L -a r a b in o sv iS , I - e a s e l and T e u c o n o s to c n e s e n t e r o i d e s .
am in o a c i d s w e re d e te r m in e d w i t h L -a r a b .in o su s
i s o l e u c i n e , v a l i n e and t h r e o n i n e .
1 7 - 5 ; g lu t a m ic a c i d ,
le u c in e ,
A r g i n i n e , p h e n y l a l o n l n e , and t y r o s i n e
w a re d e te r m in e d w i t h L a c t o b a c i l l u s e a s e l .
w ith Ia u c o n o sto c - o s e n t a - o ld c g »
T$e f o l l o w i n g
H i s t i d i n e and l y s i n e t e r e a s s a y e d
M i c r o b i o l o g i c a l v a l u e s a g r e e q u i t e tie 11
w i t h v a l u e s o b ta in e d by c h e m ic a l p r o c e d u r e s and i t
i s p o s s i b l e f o r one
a n a l y s t t o d e te r m in e a l l t h e am ino a c i d s d e s c r ib e d b y K ie r i n t h i s p a p e r
-
16
i n 4 t o 5 s a m p le s i n a p p r o x im a te ly s e v e n w o r k in g d a y s ,
(3 1 )
tte&ah&n and S n e l l
i n d i c a t e t h a t w h i l e a l l th e am ino a c i d s u se d i n t h e i r b a s a l medium
a r e n o t e s s e n t i a l f o r t h e g r o w th o f t h e o r g a n is m , h e a v i e r g r o w th i s o b t a i n ­
ed and a n t a g o n i s t i c e f f e c t s
a c i d m ix tu r e i s u s e d .
o c c u r l e s s f r e q u e n t l y when t h e c o m p le te am ino
The m ed ia u se d
in t h is
p ap er w ere v e r y s a t i s f a c t o r y
i n t h a t t h e r e w ere no i n d i c a t i o n s o f a n t a g o n i s t i c e f f e c t o b s e r v e d i n th e
v a r i o u s s a m p le s s t u d i e d .
K u ik en ( 2 4 ) h a s u sed
L a c t o b a c i l l u s a r a b ln o s u s
1 7 -5 f o r t h e d e t e r m i n a t i o n o f l e u c i n e , i s o l e u c i n e and v a l i n e and found
t h a t in c o m p le t e h y d r o l y s i s showed low v a l u e s f o r t h e s e a c i d s .
h y d r o ly s is
A l k a li n e
i s u n s a t i s f a c t o r y b e c a u s e i t b r i n g s a b o u t r a c e m ig a t io n and o n ly
n a t u r a l o c c u r r in g form s o f t h e s e a c i d s a r e u t i l i s e d .
Nyman and G o r tn e r ( 3 3 ) h ave made s t u d i e s on L a c t o b a c i l l u s a r a b i n o su s
1 7 -5 c u l t u r e s and have fou n d t h a t d e g r a d e d c u l t u r e s o f L -a r a b ln o s u s and
L - c a s e i c a n be r e j u v e n a t e d by a c h a n g e i n t r a n s f e r m edium , a g r e a t e r f r e ­
q u e n c y o f t r a n s f e r and b y i n c u b a t i o n o f th e c u l t u r e a t a n optim um te m p era ­
tu r e o f 32°C .
T hey recommend th e u s e o f l i v e r t r y p t o n e a g a r and l i v e r
t r y p t o n e b r o t h a s s t o c k c u l t u r e m e d ia .
a c id
H e g s te d
( 1 7 ) I n v e s t i g a t e d t h e am ino
r e q u ir e m e n ts o f L a c t o b a c i l l u s a r a b in o a u s 1 7 -6 and fou n d t h a t g ro w th
f a i l e d when a r i g i n i n e ,
c y s t i n e , g lu t a m ic a c i d , i s o l e u c i n e ,
le u c in e ,
m e t h io n i n e , p h e n y l a l a n i n e , tr y p to p h a n e and t y r o s i n e o r v a l i n e w ere o m itt e d
fro m t h e b a s a l m edium .
am ino a c i d s .
G rowth d id n o t o c c u r i n a m ix tu r e o f o n ly t e n
Growth m ea su rem en ts w ere made w i t h b o th t u r b i d ! m e t r i c m eas­
u r e m e n ts and b y t i t r a t i o n
o f th e l a c t i c a c id
g a v e more c o n s i s t e n t r e s u l t s .
p rod uced .
T itr a tio n u s u a lly
-
19
S t r e p t o c o c c u s f a o c a l l s and L a c t o b a c i l l u s d a l b r i l c k i l ,
S t o k e s end G unnes ( 4 2 ) h ave d e v e lo p e d a sta n d a r d a s s a y p r o c e d u r e f o r
t e n e s s e n t i a l am ino a c i d s
( i n c l u d i n g m e t h io n in e ) a p p l i c a b l e t o f o o d s t u f f
and o t h e r n a t u r a l p r o d u c t s .
E in e o f t h e am in o a e i d s c a n b e d e te r m in e d w i t h
S - i a e c a l i s and p h e n ly a la n in e w i t h L -d e X b rilch iI .
A lth o u g h tw o o rg a n ism s a r e
u se d o n ly one s ta n d a r d medium and one p r o c e d u r e i s n e c e s s a r y .
a c id
The am ino
i s m easu red by t i t r a t i o n w it h s ta n d a r d a l k a l i w i t h th e e x c e p t i o n o f
th e
h is tid in e
th e
o n ly /( l )
is o m e r s a r e a v a i l a b l e and / d ) is o m e r i s
Lower m i c r o b i o l o g i c a l v a l u e s w e re c o n s i s t e n t l y o b ta in e d
com pared w it h c h e m ic a l m e th o d s ; i t
is
in a c tiv e .
f o r m e th io n in e a s
p o s s i b l e t h a t t h e c h e m ic a l v a l u e s
may o b t a i n s u b s t a n c e s o t h e r th a n m e t h io n i n e .
Ieu o o n o eto e r e a e n t e r lo d e s t
The am Lno a c i d
b een in v e s tig a te d
r e q u ir e m e n ts o f l e u c o n o s t o c m e s e n t e r io d e e
by Dunn ( 1 1 ) .
A s ta n d a r d g ro w th c u r v e h a s b e e n d e te r m in ­
ed f o r b o th m e t h io n in e and c y s t i n e .
s a lts
P -6 0 have
He fo u n d t h a t la r g e c o n c e n t r a t i o n o f
i n h i b i t t h e g ro w th o f l e u c o n o s t o c g ie s e n t e r io d e s s o a c i d h y d r o ly s a t e s
o f p r o t e i n s h o u ld h ave m ost o f t h e a c i d rem oved b e f o r e b e i n g n e u t r a l i s e d
w i t h SaOH t o b r i n g th e pH up t o 7 ,
O th er O r g a n is m s t
Dunn ( 1 2 ) h as made d e t e r m i n a t i o n s o f m e th io n in e i n p r o t e i n h y d r o ly ­
s a t e s w ith I a c t o b a c il l u s fe r m e n t! 3 6 * .
Fe fou n d t h a t m e t h io n in e w as e s s e n ­
t i a l o r a c c e s s o r y f o r th e s a t i s f a c t o r y g r o w th o f »
L a c to b a c illu s e a s e l ,
L a c t o b a c i l l u s a r a b in o s u s 1 7 - 6 , L a c t o b a c i l l u s f e r m e n t l , and L euoo n o s to e
m e s a n t e r io d e s P - 6 0 ,
d (+ ) M e th io n in e w as fou n d t o be c o m p l e t e l y i n a c t i v e
—
20
—
In prom oting, g r o w th o f Lr-aratiI rosub and L e u o o n o sto o a e s e n t e r i o d a s *
It,
h o w ev er, s tim u la t e d t h e g ro w th o f L a c t o b a c i l l u s f e r m e n t ! t o an e x t e n t
e n t i r e l y c o a p a r a tle t o t h a t e x h i b i t e d b y t h e I ( - ) a n t i p o d e .
The a b i l i t y
cf a e d e r o o r g a n ie m t o u t i l i z e b o th o p t i c a l is o m e r s e f f e c t i v e Iy i s an
u n iq u e p r o p e r ty n o t h i t h e r t o r e c o r d e d i n th e l i t e r a t u r e ,
t h e m e th io n in e c o n t e n t o f h y d r o ly s e d c a s e i n and s i l k
o r g a n is m .
f i b r o i n w ith t h i s
The b a s a l medium C o f c o m p o s it io n g i v e n b y Lunn ( 1 0 ) was u s e d .
B c r t o n - A r ig h t ( d ) h as had v a r y s a t i s f a c t o r y r e s u l t s
0
8
h e d e te r m in e d
u s in g L -a r a b ln -
f o r f ^ic a s s a y i n g o f c y s t i n e and L su o o n o s to c mes e n t e r l o d e s f o r
m e t h io n i n e .
-
I?.
1.
21
-
SfATEMIST OF TEE PROBLEM
V a r ie t y o f ran g e g r a s s e s u sed :
( a ) A gropyron c r l s t a t u g
(b ) A ^ropyron s m l t h l l
( c ) 'fCutQloua X r a o i l l l s
2.
P r o te in s e x t r a c t e d from th e d r ie d g r a s s by means o f t
(a ) Sodium d o d e c y l s u l f a t e . S m ith (3 9 )
(b ) Form ie a c id e x t r a c t i o n , A lb an ese ( I )
Se
E x tr a c te d p r o t e i n s w ere p u r i f i e d , h y d ro ly z e d and s u b je c te d t o a n a l y s i s
f o r b o th m e th io n in e and c y s t i n e .
4.
C y s tin e e s tim a te d by Luggf s m o d if ic a tio n o f F o lin -M a re n z i method ( 2 6 ) .
6.
K e th io n ln e e s tim a te d by M oC arthy-SuI I iv a n c o l o r i m e t r i c t e s t Csonka
and D enton ( 1 0 ) .
6.
M ic r o b io lo g ic a l a s s a y s w ere c a r r i e d o u t on th e l e a f p r o t e i n e x t r a c t s
u s in g L -a ra b in o s u a f o r c y s t i n e and L eu conostoc m e se n te ro irie s f o r
m e th io n in e .
—
V.
22
—
EXPERIMENTAL * 0 »
P r e p a r a t io n o f S a m p le s«
The g r a s s sam p les w ere c o l l e c t e d from E x p erim en t S t a t i o n p l o t s lo c a te d
a t Bozeman, M o n tan a.
The g r a s s e s w ere grown u n d o r a s n e a r l y i d e n t i c a l con­
d itio n s as p o s s ib le .
The g r a s s e s w ere c u t once i n th e s p r in g and a g a in in
midsummer a f t e r th e y had re a c h e d th e e a r l y bloom s t a g e .
L i s t of G rasses and D ates o f C u t t i n g ;
A gropyron o r is ta tu m
6 -1 0 -4 6
7 -1 6 -4 6
A gropyron s m i t h l l
6 -1 0 -4 6
7 -1 6 -4 6
B o u te lo u a g r a o i l l i s
- - - -
7 -1 6 -4 6
3 -2 9 -4 6
The g r a s s e s a f t e r c u t t i n g w ere b ro u g h t t o th e l a b o r a t o r y and sp re a d
out to d ry .
They w ere d r ie d a s q u ic k ly a s p o s s i b le i n a warm room and a l l
w eeds and o th e r e x tra n e o u s m a t e r i a l ware c a r e f u l l y removed by hand p ic k in g .
A f te r d r y in g th e g r a s s e s w ere ground i n a W iley m i l l u n t i l th e y p a sse d
th ro u g h a 40-m esh s c r e e n th e n s to r e d i n a i r t i g h t g la s s j a r s .
T o ta l n i t r o g e n d e te rm in e d by th e K je ld a h l method was m u l t i p l i e d by
th e f a c t o r 6 .2 5 t o g iv e th e c ru d e p r o t e i n c o n t e n t .
p ro te in E x tra c tio n :
The f i r s t method used f o r p r o t e i n e x t r a c t i o n was t h a t o f A lbanese ( I )
w hich i s th e fo rm ic a c i d e x t r a c t i o n m ethod.
P re lim in a ry e x p e rim e n ts by
A lbanese ( I ) d e m o n s tra te d t h a t th e fo rm ic a c id e x t r a c t i o n o f f r e s h v e g e t­
a b l e s a f t e r a c e to n e e x t r a c t i o n y ie ld e d h ig h n i t r o g e n r e c o v e ry w ith an un­
a v o id a b le im p u r ity o f a p o ly s a c c h a r id e n a t u r e .
Be o b serv ed t h a t th e a d d i­
t i o n o f tw o volum es o f e t h a n o l t o one volume o f fo rm ic a c id e x t r a c t o f
23
-
s p in a c h le a v e s r e s u l t e d i n a q u a n t i t a t i v e p r e c i p i t a t i o n o f th e s e carb o h y ­
d r a t e s w ith o n ly a n e g l i g i b l e lo s s o f n i t r o g e n .
S in c e th e p o l y s a c c h a r i d e s a p p e a r t o b e t h e p r i n c i p a l c a r b o h y d r a te
c o n ta m in a n ts o f t h e f r a c t i o n s r e s u l t i n g from th e fo r m ic a c i d e x t r a c t i o n o f
v e g e ta b le s >
and t h e
A lb a n e e e h a s c o n c lu d e d t h a t t h e s o l u b i l i t y
o f th e p r o te in s
i n s o l u b i l i t y o f th e p o l y s a c c h a r i d e s i n th e fo r m ic a c i d - e t h a n o l m ix­
t u r e o f f e r s a s im p le m ethod f o r th e p u r i f i c a t i o n o f t h e p r o t e i n m o i e t y .
A lbanese s t a t e s t h a t fo rm ic a o id e x t r a c t i o n may n o t be a p p l ic a b l e t o
some ty p e s o f f e e d s t u f f , h o w ev er, i t was d e c id e d t o t r y th e method t o see
w h a t r e s u l t s c o u ld be o b ta in e d .
was used t o t e s t t h i s m eth o d .
B o u te lo u a g r a o i l l l s o u t A ugust 2 2 , 1946,
A f te r c u t t i n g , th e f r e s h g r a s s was f r o s e n
by s to r a g e In th e f r e e z i n g com partm ent o f an e l e c t r i c r e f r i g e r a t o r s e t a t
0°C .
T h is s e rv e d t o b re a k down t h e c e l l w a l l s , th e re b y in c r e a s i n g th e
p e r m e a b il ity t o s o l v e n t s . 1 1196 g . o f g r a s s was t r e a t e d in t h i s m an n er, th e n
chopped u p , p la c e d i n a f iv e g a l l o n fo o d j a r and co v ere d w ith a c e to n e f o r
two d a y s .
T h is tr e a tm e n t w ith a c e to n e te n d e d t o remove much o f th e rem ain­
in g m o is tu re and many o f th e s o lu b le p l a n t p ig m e n ts .
A f te r s ta n d in g two
d ay s th e a c e to n e was d e c a n te d o f f and th e g r a s s was w rapped i n a p ie c e o f
c h e e se c l o t h and s u b m itte d t o a c o n tin u o u s a c e to n e e x t r a c t i o n f o r 24 h o u r s .
T h is e x t r a c t i o n wa? c a r r i e d o u t a t 53°C .
S i n c e e x c e s s i v e d e c o m p o s it io n o f fo r m ic a c i d a t i t s
b o i l i n g p o in t p r e ­
v e n te d t h e u s e o f c o n t in u o u s e x t r a c t i o n t e c h n i q u e , t h e a c e t o n e e x t r a c t e d
g r a s s w as t r e a t e d a s f o l l o w s i
The c l o t h s a c k and I t s c o n t e n t s w e re rem oved
from th e e x t r a c t i o n cham ber and p la c e d i n a c u r r e n t o f a i r f o r s e v e r a l
h o u rs t o d r iv e o f f th e a c e t o n e .
A sa m p le w as th e n ta k e n o f t h e a c e t o n e e x -
“
24
—
t r a d e d g r a s s f o r a t o t a l n i t r o g e n d e te r m in a tio n and th e re m a in d e r o f th e
g r a s s was p la c e d i n 2 I . b e a k e rs co v e re d w ith 90# fo rm ic a c id and p la c e d on
a h o t p l a t e , th e te m p e r a tu r e o f w h ich was c a r e f u l l y m a in ta in e d b etw een 7 6 80°C .
The fo rm ic a c id e x t r a c t i o n was c o n tin u e d f o r 24 h o u r s , th e n re p e a te d
u n t i l n e a r l y a l l o f th e p r o t e i n and c a r b o h y d r a te m a t e r i a l had been rem oved.
A f te r th e e x t r a c t i o n was c o m p le te , th e fo rm ic a c id was d e c a n te d o f f a nd th e
f ib r o u s r e s id u e t r a n s f e r r e d t o a 2 g a l lo n enam eled p a i l and mixed m echani­
c a l l y f o r t h i r t y m in u te s w ith t h r e e l i t e r s o f 95# e th a n o l t o f a c i l i t a t e
h a n d lin g and s u b s e q u e n t d r y i n g .
T h is r e s id u e a f t e r d ry in g was l i g h t brow n
i n c o l o r and woody in a p p e a ra n c e and t e x t u r e .
A sam ple o f t h i s d r ie d
m a t e r i a l was c o l l e c t e d f o r t o t a l n it r o g e n d e t e r m in a tio n .
The a lc o h o l f i l ­
t r a t e was b ro u g h t t o volume o f 2 I . and added t o th e com bined fo rm ic a c id
f r a c t i o n s w h ich had p r e v io u s ly b een c o n c e n tr a te d i n vacuo t o I l i t e r .
The
m ix tu re th e n was a llo w e d t o s ta n d two h o u rs a t room te m p e r a tu r e and th e p r e ­
c i p i t a t e d c a rb o h y d r a te s f i l t e r e d o f f u s in g f l u t e d f i l t e r p a p e r .
The c a r ­
b o h y d ra te m a t e r i a l was d r ie d and p re s e rv e d f o r n it r o g e n d e t e r m in a tio n .
The
f i l t r a t e was re d u c e d i n v acu o t o remove a l l th e e th a n o l and fo rm ic a c i d , th e n
ta k e n t o d ry n e s s i n a la r g e e v a p o r a tin g d i s h a t a b o u t 6 0 °C .
T h is f i n a l
r e s id u e w hich c o n s t i t u t e d th e p r o t e i n m a t e r i a l was a d a rk brow n t o b la o k i n
c o lo r.
The d a rk c o l o r may have b een c a u se d by p l a n t p ig m en ts w h ich w ere n o t
removed by th e h o t a c e to n e e x t r a c t i o n o r by th e p re se n c e o f humin m a t e r i a l .
I n a n e f f o r t t o f u r t h e r p u r i f y th e p r e p a r a t i o n , i t was s u b je c te d t o h o t
a c e to n e e x t r a c t i o n f o r 24 h o u rs i n a c o n tin u o u s e x t r a c t i o n a p p a r a tu s .
T hia
l a s t e x t r a c t i o n was o f v e ry l i t t l e v a lu e and no v i s i b l e p u r i f i c a t i o n was
o b se rv e d
-
25
K x tr a o tio n o f L aaf F r o t a i n b y u se o f Sod jure D odecyl S u l f a t e , S m ith ( 3 9 ) «
The f o llo w in g g r a s s e s w ere s u b je c te d to t h i s ty p e o f e x t r a c t i o n ;
A -o ris ta tu m
A - s m ith ii
.
. . .
B -g ra e illis
.
.
.
.
.
.
.
.
......................................
. . . . . . . . . . .
5 -1 0 -4 6
. . . . . . . . .
7 -1 5 -4 6
. . .
7 -1 5 -4 6
. . . . . . . . .
3—29—46
5 -1 0 -4 6
7—16—46
The e x t r a c t i o n s w ere c a r r i e d o u t on a i r - d r i e d g r a s s e s a f t e r th e y had been
ground t o p a s s th r o u g h a 40-m esh s i e v e .
H e p r e o ip ita te d sodium d o d e e y l s u l f a t e was u sed f o r t h i s e x t r a c t i o n .
A sam ple o f 50 g . was p la c e d i n a g l a s s j a r and a b o u t 200 c o . o f a 2$ s o lu ­
t i o n o f sodium d o d e e y l s u l f a t e added u n t i l th e j a r was a b o u t o n e - h a l f f u l l .
The j a r was p la c e d i n a s h a k in g m achine i n a h o r i z o n t a l p o s i t i o n to g iv e
th e maximum s w i r l i n g m o tio n , and sh a k e n f o r th r e e h o u r s .
The j a r was th e n
removed and th e m a t e r i a l f i l t e r e d th r o u g h a p ie c e o f c h e e se c l o t h sq u e e z in g
o u t th e e x c e s s l i q u i d .
T h is e x t r a c t i o n was c a r r i e d o u t t h r e e t i i r e s f o r 3
h o u rs e a c h u s in g f r e s h sodium d o d e e y l s u l f a t e s o l u t i o n eac h ti m e .
The f i l ­
t r a t e was c e n t r if u g e d a f t e r e a c h e x t r a c t i o n and th e s lu d g e th u s o b ta in e d
was s u b m itte d t o f u r t h e r e x t r a c t i o n .
A f t e r th e f i n a l e x t r a c t i o n , 96;% e t h a n o l
was added w ith r a p id s t i r r i n g t o th e c l e a r f i l t r a t e s and th e n allo w ed t o
s ta n d u n t i l p r e c i p i t a t i o n was c o m p le te .
The s u p e r n a ta n t l i q u i d was d e c a n te d
o f f and th e p r o t e i n m a t e r i a l was p u r i f i e d by a c o n tin u o u s a c e to n e e x t r a c t i o n
f o r 24 h o u r s .
A f te r th e a c e to n e e x t r a c t i o n th e p r o t e i n p r e p a r a t io n was p la c e d i n a
vacuum oven a t 5 0 °C . f o r 3 h o u rs u n t i l th e m a t e r i a l was th o ro u g h ly d r y .
The
f i n a l p ro d u c t was th e n ground up i n a m o r ta r t o f a c i l i t a t e homogeneous sam pl­
in g .
The p ro d u c t was w h ite t o l i g h t g ra y i n c o l o r w ith a s l i g h t g r e e n is h
**
tin t.
26
*
K je ld a h l n i t r o g e n d e te r m in a tio n s w ere ru e o n 2
( a ) o r i g i n a l g r a s s sam ple
(b ) s lu d g e re m a in in g from c e n t r i f u g i n g
(0 ) r e s id u e a f t e r e x t r a c t i o n w ith sodium d o d e c y l s u l f a t e
(d ) p u r if ie d p r o te in .
H y d ro ly s is o f h r o t e i n :
I n view o f th e s a t i s f a c t o r y r e s u l t s o b ta in e d by B a rto n -W rig h t ( 8 ) ,
S c h w e ig e rt (3 7 ) and S to k e s (4 2 ) u s in g d i l u t e s o l u t i o n s o f B C l, i t was d e ­
c id e d t o u se th e f o llo w in g p r o c e d u r e ;
One gram sam ple o f f i n e l y ground
m a t e r i a l was h y d ro ly s e d by a u to c la v in g f o r s i x h o u rs a t 120°C and 15 pounds
p r e s s u r e w ith 26 m l. o f 2 .6 B HC1.
f l a s k s f i t t e d w ith g la s s c a p s .
Tiie h y d r o ly s is was c a r r i e d o u t in 50 m l.
A f te r h y d r o ly s is th e s o l u t i o n was allo w ed t o
c o o l and th e pH a d j u s t e d t o 6 .8 u s in g th e Beckman pH m e te r .
The s o lu ti o n s
w ere th e n b ro u g h t t o a volume o f 100 m l ., f i l t e r e d , co v e re d w ith a t h i n l a y e r
o f s u l f u r f r e e to lu e n e and s to r e d i n th e r e f r i g e r a t o r .
A ll th e l e a f p r o te i n s
a s w e ll a s c a s e i n s a m p le s , w h ich w ere u sed to ch ec k th e d i f f e r e n t a s s a y
m e th o d s, were h y d ro ly s e d by t h i s m eth o d .
,
C o lo r im e tr ic Ketiiod f o r C y s tin e , F o lln and K a re n s! ( 1 4 ) ; Iu g g ( 2 6 ) ; K a s se l
and Frand ( 2 2 ) .
T h e o ry ;
F h o sp h o -1 8 -T u n g s tic a c id ( F b l i n t S r e a g e n t) was u sed f o r th e c o l o r i ­
m e tr ic d e t e r m in a tio n o f c y s t i n e .
The b lu e c o l o r d e v elo p ed depends upon th e
r e d u c t io n o f p h o sp h o -18- t u n g s t i c a c id b y c y s t e i n e .
by sodium s u l f i t e t o g iv e c y ste in e .
C y s tin e i s f i r s t red u ced
The p h o s p h o - 1 8 - tu n g s tic a c id is p re p a re d
e s s e n t i a l l y by t r e a t i n g sodium t u n g s t a t e w ith p h o sp h o ric a c i d .
The o o lo r a -
27
-
t i o n d ev e lo p e d by a r e d u c e r may v a ry w ith th e tim e a llo w e d f o r th e r e a c t i o n ,
th e c o n c e n tr a ti o n and am ounts o f th e r e a c t i n g m a t e r i a l s , th e pH o f th e r e a c ­
t i n g m ix tu r e , s a l t c o n c e n tr a ti o n and th e ty p e and o r d e r i n w h ich th e m a te r­
ia ls a re added.
E v id en ce h as b een adduced by Lugg (2 6 ) t o show t h a t th e r e a c t i o n b e ­
tw een c y s ti n e and s u l p h i t e s i n c o ld aqueous s o l u t i o n i s n o t a r e d u c tio n i n
th e o r d in a r y s e n s e o f th e te r m , b u t t h a t i t i s m ost p ro b a b ly e x p re s s e d by
th e e q u a tio n I i - S - S - R
+ H2SO3 ---------►
FSH + '!HSgO3 w here % - S - S - R
and R S H r e p r e s e n t c y s t i n e and c y s te i n e r e s p e c t i v e l y .
2 Ti
H + re a g e n t
...... !I..*.
R - S - S - R r
Then
red u ced r e a g e n t.
By e s ti m a t in g
c y s te i n e and c y s ti n e i n a c id s o l u t i o n a l l th e d is a d v a n ta g e s o f a l k a l i n e
c o l o r developm en t may be a v o id e d .
The d is a d v a n ta g e s o f a c id c o l o r d ev elo p m en t in c lu d e s l i g h t lo s s in
c o l o r and d im in u tio n in th e r a t e o f d e v e lo p m e n t.
F o r a l l aro u n d u t i l i t y a
pH o f 6 .7 h a s b een s e l e c t e d , and t o m in im ise c o l o r d ev elo p m en t by e x tra n e o u s
re d u c e rs and f o r th e sak e o f economy o n ly s m a ll am ounts o f r e a g e n t a r e em­
p lo y e d .
A s ta n d a r d i s p re p a re d u s in g c y s t i n e p lu s s u l f i t e .
r e d u c e rs a r e p r e s e n t t h e i r e f f e c t s m ust be found by a r t i f i c e .
When e x tra n e o u s
I n th e p re s e n c e
o f HgClg c y s te i n e w i l l g iv e no c o l o r , th u s th e b la n k u sed f o r co m p ariso n was
p re p a re d e x a c t l y th e same a s th e unknown e x c e p t f o r t h e a d d i t i o n o f HgClg
w hich rem oves t i n c y s te i n e le a v in g o n ly e x tra n e o u s r e d u c e rs t o e f f e c t c o l o r
d e v e lo p m e n t.
Lugg’ s (2 6 ) m o d if ic a tio n o f th e F o lin and l a r e n s i method (14) i s d e s ig n ­
ed f o r use w ith th e v i s u a l c o lo r im e te r and he has ad o p te d a r a t h e r in g e n io u s
method o f c o r r e c t i n g f o r e x tra n e o u s r e d u c e r s .
The c o r r e c t i o n f o r e x tra n e o u s
**■
26
*
re d u c e rs may be v e ry g r e a t l y s i m p l i f i e d by th e u se o f th e s p e c tr o p h o to m e te r .
K a s s e ll and Brand (22) have a d a p te d Lugg1S - F e lin m ethod t o th e use o f
th e P u l f r i o h P h o to m e te r.
P erh ap s th e method u sed i n t h i s t h e s i s sh o u ld
more c o r r e c t l y be r e f e r r e d t o a s th e K a s s e l and Brand r a t h e r th a n th e Lugg
m ethod, a lth o u g h th e y a r e e s s e n t i a l l y th e same from th e s ta n d p o i n t o f th e
r e a g e n ts used t o b r i n g a b o u t t h e c o l o r d e v e lo p m e n t.
R e a g e n ts :
A ll r e a g e n ts used -were th e same a s th o s e s p e c i f i e d b y Lugg ( 2 6 ) .
F h o s p h o - ia-t u n g s tI c A c id R e a g e n t:
T h is e a s p re p a re d a c c o r d in g t o th e d i r e c t i o n s o f F o lin and M arenxi (4 0 )
w i t h th e s e p a r a t i o n o f any molybdenum p r e s e n t a c c o rd in g t o t h e i r p l a n .
may be looked upon a s c o n t a in in g p e r l i t e r 100 g . o f th e s a l t
172 grains o f HgPO4 and 1 6 .1 g . o f Li OH.
It
Ka2WO4 * 2ligO,
The sodium t u n g s t a t e sh o u ld be p r e ­
p a re d by d i s s o l v i n g th e n e c e s s a r y am ount o f p r e c i p i t a t e d WOg i n th e n e c e s s a r y
amount o f KaCH s o l u t i o n .
I f th e r e a g e n t f a i l s t o com ply w ith th e f o llo w in g
re q u ire m e n ts i t m u st be d is c a r d e d *
(1 )
I t m ust g iv e no c o l o r a t i o n w ith t y r o s i n e o r try p to p h a n e a t
any pH.
(2 )
At pH 6 .7 and 8 . 4 , 4 mg. o f c y s t i n e i n th e p re s e n c e o f 2 c e .
o f r e a g e n t and I m illim o le o f s u l p h i t e m ust g iv e n o t le s s th a n
2 .9 6 tim e s th e c o l o r a t i o n d ev e lo p e d by I mg. o f c y s t i n e .
(S )
At pH 6 .7 and 8 .4 and i n th e a b s e n c e o f s in e and ammonium
s a l t s , 80 mg. o f c y s t i n e w ith 2 c c . o f r e a g e n t m ust g iv e
a lm o s t e x a c t l y se v e n tim e s th e c o l o r a t i o n d ev elo p ed by 2 mg.
o f c y s tin e .
29
-
The r e a g e n t p re p a re d by th e f o llo w in g method met a l l th e s e re q u ire m e n ts i
60 grams o f sodium t u n g s t a t e and 100 c c . o f w a te r were tpdded t o a 600 e c .
F lo re n c e f l a s k , sh ak en u n t i l th e t u n g s t a t e d i s s o l v e d , th e n added slo w ly w ith
c o o lin g 10 m l. o f 86% p h o s p h o ric a c i d .
The s o l u t i o n m ust n o t be allo w ed t o
become w arn from t h e h e a t o f r e a c t i o n .
H ydrogen s u l f i d e was p assed i n t o th e
p h o sp h o tui g s t i o a c id s o l u t i o n a t a v e r y m o d erate r a t e f o r 20 m in u te s .
At
th e end o f th e f i r s t 3 -4 m in u te s 6 m l. o f HgPO* was added w ith o u t i n t e r r u p t ­
in g th e HgS c u r r e n t .
(T h is e n a b le s one to o b ta in th e molybdenum s u l f i d e i n a
somewhat l e s s f i n e l y d iv id e d c o n d i tio n so I t can be f i l t e r e d more e a s i l y . )
A t t h e end o f 20 m in u te s th e s o l u t i o n was f i l t e r e d u s in g a Ho. 40 Whatman
f i l t e r p a p e r.
When c o n d i tio n s a r e r i g h t , th e f i l t r a t e sh o u ld be c l e a r and
have g r e e n i s h c o l o r .
The f i l t r a t e was th e n t r a n s f e r r e d t o a s e p a r a t o r y fu n ­
n e l and 150 e e . o f 96% e t h a n o l added w ith s h a k in g .
The b o tto m s o l u t i o n con­
t a i n e d a l l th e p h o sp h o tu n g at i c a c id i n a s u p e r s a tu r a t e d s o l u t i o n .
s o l u t i o n was th e n w ith d raw n
i n t o a w eighed 500 m l. f l a s k
th e w e ig h t o f t h e c o n t e n ts was 150 g ram s.
The
T his
and w a te r added u n t i l
s o l u t i o n was b o il e d a few m in­
u te s o v e r a m icro b u r n e r and t e s t e d w ith le a d a c e t a t e p a p e r t o be s u r e a l l th e
h y d ro g en s u l f i d e had b e e n rem oved.
p h o s p h o ric a c id was a d d e d .
f o r one h o u r .
The flam e was c u t down and 10 m l. o f 86%
The f l a s k was th e n b o ile d g e n t l y w ith r e f lu x i n g
Tl# s o l u t i o n
was f i l t e r e d and a few d ro p s
o f brom ine w ere added
and th e s o l u t i o n was b o ile d
u r t i l a l l th e b lu e c o lo r was
g o n e , th e n b o ile d
v ig o r o u s ly s e v e r a l m in u te s t o remove th e b ro m in e .
w ith a b e a k e r and c o o le d u n d e r ru n n in g w a te r .
The f l a s k was th en co v e re d
A li th iu m p h o sp h a te s o l u t i o n
was p re p a re d by p la c in g 1 2 .5 g . o f li th iu m c a rb o n a te and 25 m l. o f p h o sp h o r­
i c a c id in a l i t e r L e a k e r , th e n s lo w ly a d d in g 126 m l. o f w a t e r , th e r e s u l t i n g
s o l u t i o n was th e n b o il e d t o remove CC#, c o o le d , and th e n added t o th e p h o s -
—
50
—
p h o tu n g s tic a o id s o l u t i o n and th e t o t a l volum e b ro u g h t t o 600 m l. w ith d i s ­
t i l l e d w a te r.
Sodium A c e ta te S o l u t i o n ;
A 4 m o la r sodium a c e t a t e s o l u t i o n was p re p a re d c o n ta in in g 644 g . o f
th e s a l t CgHgOgSaeSEgO p e r l i t e r o f s o l u t i o n .
C i t r a t e B u ffe r S o l u t i o n ;
T h is c o n ta in s 106 g . o f c r y s t a l l i n e c i t r i c a c id CgHg OyeBgO, 6 2 .6 g .
o f NaOH, 1 3 .6 g . o f ZnClg and 2 6 .8 grama o f BH4CI p er l i t e r .
I t was p r e ­
p a re d by s h a k in g th e c i t r i c a o id and NaOH w ith ab o u t 600 c c . o f w a te r w ith
c o n s ta n t c o o lin g u r t i l e n t i r e l y d i s s o l v e d .
The ZnClg was d is s o lv e d i n 20 c o .
o f w a te r and added t o th e co ld s o l u t i o n , and t h i s fo llo w e d by th e NH4 Cl d i s ­
s o lv e d i n a b o u t 200 c c . o f w a te r .
The m ix tu re th e n d i l u t e d t o I l i t e r .
B u ffe re d S u lp h it e S o l u t i o n ;
9 .6 grams o f sodium m e ta b is u lp h ite (NagSgO5 ) d is s o lv e d i n a b o u t 70
c c . o f w a te r , 15 c c . o f 4 K sodium a c e t a t e s o l u t i o n was added and th e mix­
t u r e d i l u t e d to 100 c o .
Lugg s t a t e s t h a t t h i s s o l u t i o n w i l l keep f o r m onths
i n a s to p p e re d b o t t l e , h o w ev er, i t was found n e c e s s a iy to p r e p a r e t h i s s o lu ­
t i o n f r e s h d a l l y i n o r d e r t o o b ta in a r e l i a b l e s ta n d a r d c u r v e .
B e ro u rlo c h l o r i d e 1
A 0 .1 N s o l u t i o n o f m e rc u ric c h l o r i d e was p re p a re d c o n t a in in g 2.72
grams o f HgClg p e r 100 0 0 . o f s o l u t i o n .
S ta n d a rd C y s tin e S o l u t i o n ;
0 .5 gram s o f I ( - ) c y s t i n e was d is s o lv e d i n 60 o c . o f I N BCl and d i l -
-
31
a te d t o 600 o o . t o p ro v id e a .1 N HCl s o l u t i o n c o n t a in in g I mg. o f c y s tin e
p e r m l.
The s o l u t i o n was c o v e re d w ith a t h i n la y e r o f to lu e n e and s to r e d
i n tlie r e f r i g e r a t o r .
S o lu ti o n was d i l u t e d I il O t o g iv e .1 mg. o f c y s tin e
p e r m l. when u s e d .
E s tim a tio n in Q e n e r a ls
A ll e s ti m a t io n s w ere c a r r i e d o u t i n s ta n d a r d 100 c o . f l a s k s .
was p re p a re d a t th e same tim e a s unknown.
S ta n d a rd
S even m in u te s w ere allo w ed f o r
th e c o l o r d e v e lo p m e n t, th e n th e s o l u t i o n was d i l u t e d t o th e m ark and read
a t 660 mu. in a Coleman s p e c tr o p h o to m e te r .
A liq u o ts o f th e unknown c y s t i n e
s o l u t i o n w ere b ro u g h t t o a pH o f 5 .7 w ith sodium a c e t a t e s o l u t i o n .
P r e lim in a r y
w ork was done to d e te rm in e w hat e f f e c t th e o r d e r o f
a d d i t i o n o f th e r e a g e n ts had on th e c o l o r d ev elo p m en t and s ta n d a r d c u rv e s
th u s o b ta in e d .
The r a t e o f c o l o r d ev elo p m en t was s t u d i e d .
u s in g f r e s h s u l p h i t e r e a g e n t was i n v e s t i g a t e d .
H y d ro ly sed c a s e i n sam ples
a s w e l l a s l e a f p r o t e i n s w ere a s sa y e d by t h i s m ethod.
t o p ro v id e l i t e r a t u r e c h e e k s .
The e f f e c t o f
The c a s e i n was used
V ita m in t e s t c a s e i n was f i r s t u s e d , how ever,
a f t e r a number o f a s s a y s i t was found t o be u n s a t i s f a c t o r y and c a s e in p r e ­
p a re d by th e H a m e rste in m ethod proved t o be o f b e t t e r q u a l i t y th a n th e com­
m e r c ia l p r o d u c t.
A ssays w ere ru n w ith c y s t i n e added t o the sam ple b o th b e ­
f o r e anti a f t e r h y d r o ly s is t o d e te rm in e c y s t i n e re c o v e re d .
E x p e rim e n ta l P ro c e d u re s i
The o rd e r o f a d d i t i o n o f th e r e a g e n ts was found t o be v e ry im p o r ta n t.
The r e a g e n ts w ere added from a b u r e t t e t o th e r e a c t i o n f l a s k , th e f l a s k
w e ll sh ak en a f t e r eac h a d d i t i o n .
The on ly o r d e r g iv e n by Lugg (2 6 ) was fo u n d
-
52
t o be s a t i s f a c t o r y and a n y ch a n g e i n th e o r d e r o f t h e a d d i t i o n o f th e r e a g e n t s
r e s u lte d
i n u n s a t i s f a c t o r y c o l o r d e v e lo p m e n t and I n c o n s i s t e n c y i n th e s t a n ­
d ard c u r v e s o b t a i n e d •
To o b t a i n a sta n d a r d c u r v e th e f o l l o w i n g r e a g e n t s w e re
ad d ed i n ^ iv e n o r d e r t o a 100 m l* v o l u m e t r ic f l a s k w i t h s h a k in g a f t e r e a c h
a d d itio n .
10 c o . o f c i t r a t e b u f f e r
2 o o . p h o s p h o -1 8 -tu n g s tic a c id
reagen t
2 c c . sod iu m a c e t a t e
S ta n d a rd c y s t i n e s o l u t i o n ( . 1 mg. / m l . )
I o c . b u ffe r e d s u lp h ite
The c o l o r w as a llo w e d t o d e v e l o p 7 m in u te s and t h e f l a s k d i l u t e d t o th e
m ark ,
h s ta n d a r d b la n k was p r e p a r e d same a s a b o v e o m i t t i n g t h e c y s t i n e s o l u ­
tio n .
A s ta n d a r d b la n k w as fou n d n e c e s s a r y inasm uch a s t h e s u l p h i t e
t e n d s t o r e d u c e th e p h o sp h o tu n g s t i c
a c id .
reagent
The am ount o f c o l o r due t o th e
r e d u c t i o n d e p e n d s upon t h e a g e o f t h e s u l p h i t e s o l u t i o n .
s ta n d a r d c u r v e w as from 0 . 1 m g. t o 0 . 8 m g. o f c y s t i n e .
o f c o n c e n t r a t i o n w ere d e te r m in e d f o r th e s ta n d a r d c u r v e .
The ra n g e o f th e
At f i r s t 8 le v e ls
L a te r , h ow ever,
i t w as fou n d t h a t f o u r p o i n t s w ere s u f f i c i e n t t o p r o v id e r e l i a b l e a c c u r a c y .
S ta n d a r d c y s t i n e w as run a t 0 . 1 , 0 . 2 , 0 . 4 , and 0 . 8 m g. o f c y s t i n e w it h r e ­
l i a b l e u n ifo r m ity in th e r e s u l t s .
A s ta n d a r d c y s t i n e c u r v e i s
g iv e n on Page
54» F ig u r e 2 .
I n o r d e r t o d e te r m in e th e r a t e o f c o l o r d e v e lo p m e n t t h e f o l l o w i n g
sch em e w as u sed i
U s in g 10 m l. o f s ta n d a r d c y s t i n e and t h e o r d e r o f a d d i t i o n
i n d i c a t e d a b o v e , t h e c o l o r was a llo w e d t o d e v e lo p f o u r m i n u t e s , th e n 2 c l .
a l i q u o t s w e re t a k e n e v e r y m in u te f o r s e v e n m i n u t e s .
Each a l i q u o t was lm m ed-
-
58
l a t e l y d i l u t e d to 3 a.1. w ith d i s t i l l e d w a te r t o h a l t th e e o l o r dev elo p m en t
and re a d a t 650 mu.
A f te r th e f i r s t r e a d in g th e tu b e s w ere allo w ed t o s ta n d
50 m in u te s , th e n read a g a in *
o c c u r a t 7 m in u te s •
The optimum c o l o r d ev elo p m en t was found t o
The d i l u t i o n h a l te d th e c o l o r r e a c t i o n and t h e r e was no
change i n th e re a d in g s ta k e n a t th e end o f th e 30 m in u te p e r i o d .
A cu rv e
i l l u s t r a t i n g th e r a t e o f c o l o r d ev elo p m en t i s g iv e n on Page 5 3 , F ig u re I .
I t h a s b een th o u g h t a d v is a b le by some i n v e s t i g a t o r s t o d e c o l o r i s e th e p r o ­
t e i n h y d r o ly s a te b e f o r e t e s t i n g f o r c y s t i n e , v a r io u s d e c o l o r i s i n g c h a r c o a ls
a r e recommended f o r t h i s p u rp o s e .
S e v e r a l a d s o rb a n t c h a r c o a ls w ere t e s t e d and found t o be u m a t i s f a c t o r y
f o r t h i s p u rp o se s in c e in a d d i t i o n t o d e c o l o r i z i n g th e h y d r o ly s a t e , a p r a c ­
t i c a l l y q u a n t i t a t i v e a d s o r p ti o n o f c y s t i n e was o b s e r v e d .
300 mg. o f D arco
was added to 10 s I . o f s ta n d a r d c y s ti n e s o l u t i o n , th e m ix tu re sh ak en and a l ­
lowed t o s ta n d f o r I h o u r , f i l t e r e d and th e c y s t i n e d e te rm in e d on th e f i l ­
tra te *
B8% o f th e c y s t i n e was a d s o r b e d .
T h is was t r i e d w ith s e v e r a l o th e r
c h a r c o a ls w hich w ere a v a i l a b l e and a l l showed s i m i l a r r e s u l t s .
So in view
o f th e s e o b s e r v a t i o n s , i t was d e c id e d a d v is a b le t o f o r e g o th e d e e o l o r i s a t i o n .
The h y d r o ly s a te s ran g ed from l i g h t amber t o b r o w i n c o l o r and on d i l u t i o n
o f th e a l i q u o t sam ple t o 100 m l. t h i s c o l o r was g r e a t l y d im in is h e d .
By u s in g
th e f o llo w in g scheme f o r p r e p a r in g th e unknown b la n k c o r r e c t i o n i s reads f o r
th e c o l o r o f o r i g i n a l s o l u t i o n a s w e ll a s t h a t due t o e x tr a n e o u s r e d u c e r s .
Unknown B lan k
Unknown
10 c c . c i t r a t e b u f f e r
10 c c . c i t r a t e b u f f e r
2 c o . c o lo r re a g e n t
2 c c . c o lo r re a g e n t
2 c c . sodium a c e t a t e
2 o o . sodium a c e t a t e
(C o n tin u ed on page 5 4 ) .
%
—
54
—
(C o n tin u ed from page S 3 ) .
Unknown
Unknown B lank
A liq u o t o f unknown ( t i t r a t e d t o pH 5 .7 w ith
sodium a c e t a t e )
I c c . b u ffe re d s u lp h ite
I e c . m e rc u ric c h lo r id e
a l i q u o t ok uukuauin (pH 6 .7 )
I c c . b u ffe re d s u lp h ite
eleven m in u te s was th e tim e allo w ed f o r c o l o r d ev elo p m en t and th e volume was
th e n d i l u t e d t o t h e 100 m l. m ark .
The same e l s e a l i q u o t i s ta k e n f o r t h e un­
known and th e b la n k so t h a t b o th c o n t a in th e same ooncen' r a t i o n o f e x tra n e o u s
re d u c e rs.
The 100 m l. f l a s k c o n ta in in g th e r e a c t i o n m ix tu re was th o ro u g h ly
m ixed and a l i q u o t s t r a n s f e r r e d t o c a l i b r a t e d c o lo r im e te r t u b e s , th e re a d in g s
ta k e n a t 650 mu.
The unknown b la n k was p la c e d in th e c o l o r i m e t e r , th e s c a le in d i c a t i n g
th e p e r c e n t t r a n s m is s i o n was s e t a t z e r o and th e unknown s o l u t i o n th e n m at­
ched a g a i n s t t h i s b la n k .
The amount o f c y s t i n e was th e n c a l c u l a t e d from th e
p e r c e n t t r a n s m is s i o n w ith r e f e r e n c e t o th e s ta n d a r d c u r v e .
I t was found t h a t th e age o f th e s u l p h i t e r e a g e n t g r e a t l y e f f e c t e d th e
s lo p e o f t h e s ta n d a r d cu rv e and t h a t by u s in g f r e s h l y p re p a re d s u l p h i t e s o l u ­
t i o n p r a c t i c a l l y th e same c u rv e was o b ta in e d ea c h ti m e .
C o lo r im e tr ic D e te rm in a tio n o f M eth io n in e u s in g Sodium N itr o p r u s a id e 'e a g e n t .
(so), (16), (ioT:
-------------
E x p e rim e n ta l P ro c e d u re s i
An a l i q u o t o f a c id h y d ro ly z e d p r o t e i n was t r a n s f e r r e d q u a n t i t a t i v e l y to
a 26 m l. v o lu m e tr ic f l a s k and s u f f i c i e n t c o n e . ECl added t o b r i n g th e a c id
c o n c e n tr a ti o n up to 4—5/' HCl (w hich i s a s u i t a b l e c o n c e n tr a ti o n f o r th e p r e ­
c i p i t a t i o n o f th e b a s ic amino a c id s w ith p h o s p h o tu n g s tic a c i d ) .
An aqueous
s o l u t i o n o f p h o sp h o tu n g s tIo a c id c o n ta in in g I gran p e r I m l. was added d ro p -
w is« U L til p r e c i p i t a t i o n was c o m p le te , th e n a few d ro p s i n e x c e s s .
S o lu tio n
was th e n oad# t o a volume o f 26 e c . w ith d i s t i l l e d w a te r , allo w ed t o s ta n d in
an io e b a t h f o r two h o u r s , a f t e r w hich i t was c e n tr if u g e d f o r t e n m in u tes and
p la c e d in th e I c e w a te r b a th a g a in f o r o n e - h a l f h o u r .
was th e n f i l t e r e d th r o u g h a number 40 Whatman p a p e r .
The s u p e r n a ta n t li q u i d
A f te r th e f i l t r a t e had
re a c h e d room te m p e r a tu r e , a n a l i q u o t c o n t a in in g n o t more th a n 300 mg, o f th e
o r i g i n a l p r o t e i n m a t e r i a l was t r a n s f e r r e d t o a n o th e r 25 m l. v o lu m e tr ic f l a s k
and 6 N SaQH added d ro p w ise t o a pH o f 4 u s in g th e Beckman pE m e te r .
The
s o l u t i o n was th e n made t o volum e w ith d i s t i l l e d w a te r .
An a l i q u o t o f 4 m l. o r l e s s o f t h i s f i l t r a t e was t r a n s f e r r e d t o a cen ­
tr if u g e tu b e .
To t h i s 2 m l. o f 6 N NaDH was added and s u f f i c i e n t q u a n t i t y
o f d i s t i l l e d w a te r t o b r in g th e t o t a l volume t o 6 m l.
p re p a re d
Then I m l. o f f r e s h l y
sodium n i t r o p r u s s i d e was added and th e c o n te n ts o f th e tu b e
th o ro u g h ly s t i r r e d .
The tu b e was p la c e d i n a w a te r b a th f o r 8 m in u tes a t
4 0 ° C ., th e n In a n ic e w a te r b a th f o r 6 m in u te s a f t e r w h ich 2 .6 m l. o f 2 0 ''
HCl was added s lo w ly w ith v ig o ro u s s h a k in g .
The s h a k in g was c o n tin u e d i n
th e b a th u n t i l th e oi a - e t e r i s t i s c o lo r d ev elo p m en t i n a b o u t I m in u te , th e n
s t i r r e d m e c h a n ic a lly f o r a n o t h e r m inute a t room te m p e r a tu r e .
The s o l u t i o n
a t t h i s p o in t o f te n becom es s l i g h t l y tu r b i d so i t was fo u n d n e c e s s a r y t o
c e n t r i f u g e a t h ig h speed f o r 6 m in u te s and th e n t r a n s f e r th e s o l u t i o n to a
c a l i b r a t e d c o l o r i m e t e r tu b e and read a t 610 mu i n th e Coleman s p e c tro p h o ­
to m e te r .
A s ta n d a r d m e th io n in e cu rv e was o b ta in e d by t r e a t i n g a s ta n d a r d s o lu ­
t i o n i n ( . 1 mg. o f m e th io n in e p e r m l .)
e x a c tly th e same m an n er.
D is tille d
w a te r was used f o r th e b la n k and th e c u rv e p l o t t e d w ith lo g o f p e r c e n t
tr a n s m is s i o n v e . mg. o f m e th io n in e .
The ra n g e o f th e c u rv e was from .2 6 mg.
—
56
—
t o 1*25 mg. o f m e th io n in e w ith re a d in g s ta k e n a t 6 l e v e l s .
By u s in g f r e s h l y
p re p a re d sodium n l t r o p r u s e i d e re a g e n t t h e c u rv e was found t o be e x a c t l y th e
same e a c h tim e ( w ith in l i m i t s o f e x p e r im e n ta l e r r o r ) w ith th e s lo p e o f th e
c u rv e rem a in in g c o n s t a n t .
F ig u re 6 .
A s ta n d a r d m e th io n in e cu rv e i s g iv e n on Page 6 7 ,
The m e th io n in e c o n te n t o f a c id h y d ro ly s e d l e a f p r o t e i n s , v ita m in
t e s t c a s e i n , and f r e s h l y p re p a re d c a s e i n was d e te rm in e d by t h i s m ethod.
M ic ro b io lc , l e a l D e te rm in a tio n o f C y s tin e U sinv L a c to b a o l llu s - a r a b ln o s u s 1 7 -5 ,
A ll m ic ro o rg a n ism s used f o r th e s e t e s t s w ore o b ta in e d d i r e c t l y from
A m erican Type C u ltu r e C o l l e c t i o n ,
W ashington 0 . C.
The g r e a t e s t c a r e was
ta k e n i n h a n d lin g th e s to c k c u l t u r e s to p r e v e n t any c o n ta m in a tio n .
Im m ediate­
ly upon r e c e iv in g th e c u l t u r e s , t r a n s f e r s w ere made t o l i v e r - t r y p t o n e - a g a r s ta b s
w ith w eekly t r a n s f e r s b e in g made from th e a g a r s ta b t o Xjtvaie k r y p to n s b r o t h ,
th e n t o t h e a g a r a g a i n th e f o llo w in g w eek.
T h is p ro c e s s was r e p e a te d e v e ry 7
d ay s a s recommended by Fyman and G e r tn e r (35) f o r in c r e a s in g th e a c t i v i t y o f
L. a ra b in o a u s 1 7 -5 .
A s i m i l a r medium p re p a re d from b a o to p ep to n e and b a e to t r y -
p tc n e w ith r i b o f l a v i n added was found t o be ev en more e f f e c t i v e th a n th e l i v e r t r y p t o e e medium in s t i m u l a t i n g th e g row th o f I,. a r a b in o s u s 17-6 and L euconostoc
m e s e n te r io ld e s P -6 0 .
A ll amino a c id s and v ita m in s used in p r e p a r in g th e s ta n d a r d m edia w ere o f
th e h i g h e s t p u r i t y o b ta in a b le and w here p o s s i b le th e d l s y n t h e t i c amino a c id s
w ere used r a t h e r th a n n a t u r a l o c c u r r in g p r o d u c t b eca u se o f th e h ig h e r p u r i t y
o f th e s y n t h e t i c p r e p a r a t i o n s .
W ater used f o r m aking up th e s y n t h e t i c m edia was d i s t i l l e d
t.la s a t o e l i m i n a t e an y e f f e c t due t o heavy m e ta l i o n s .
from p y rex
The r e d i s t i l l e d w a te r
was s to r e d i n a 5 g a l lo n p y rex g l a s s b o t t l e and t i g h t l y c o v e re d t o keep o u t d u s t .
-
37
-
A ll g la s s w a r e u sed was c le a n e d e i t h e r w ith D r e f t o r tr is o d iu m p h o sp h a te
and th o ro u g h ly r in s e d w ith d i s t i l l e d w a t e r .
Chromic a c id c le a n in g s o l u t i o n
was av o id ed t o p re v e n t any e f f e c t due t o ch ro m ate i o n s .
A ll t r a n s f e r s w ere
c a r r i e d o u t in a n i n o c u l a t i n g room w here d u s t and a i r c u r r e n t s w ere k e p t a t
a minimum t o p r e v e n t c o n ta m in a tio n by s p o re fo rm in g o rg a n is m s .
Tlie s ta n d a r d zned iumro commended by B a rt o n - b r ig h t (5 8 ) was used w ith
c e r t a i n m o d if ic a tio n # w hich w ere found t o oe n e c e s s a r y .
P r e p a r a tio n o f S to c k S o l u t i o n s i
The s to c k s o l u t i o n s d e s c r ib e d below w ere p re s e rv e d in t h e p re s e n c e o f
a t h i n la y e r of s u lp h u r f r e e to lu e n e and s t o r e d i n a r e f r i g e r a t o r .
A ll s to c k
s o l u t i o n s w ere p re p a re d w ith g la s s d i s t i l l e d w a t e r .
1 - c y s t Ine :
0 .6 grams o f I - c y s t i n e was d is s o lv e d in 500 m l. o f w a te r
and 2 m l. o f c o n c e n tr a te d BCl was a d d e d , th e f i n a l volume made up to 500 m l.
and s t o r e d i n a r e f r i g e r a t o r .
T h is c y s t i n e s o l u t i o n was d i l u t e d b e fo re u se
t o g iv e a s ta n d a r d s o l u t i o n c o n ta in in g 10 m icro -g ram s o f c y s t i n e p e r m l.
G lu c o s e , sodium a c e t a t e , sodium c h l o r i d e and ammonium s u lp h a te were
w eighed o u t a s r e q u i r e d .
B io tin t
An am poule c o n t a in in g 25 u g . o f th e f r e e a o id was made up
t o 250 m l. w ith w a te r .
up t o v olum e.
One m l. o f s a l t s o l u t i o n A w a s added p r i o r t o m aking
The s o l u t i o n c o n ta in e d 0 .1 u g . o f B i o t i n p e r m l.
p -a m ln o b e n to io a c i d ;
0 .1 gram o f p -am in o b en zo io a c id was d is s o lv e d in
100 m l. o f w a te r c o n ta in in g I m l. o f g l a c i a l a c e t i c a c i d .
—
38
•
T h iam io e, B l e o t l n i e s o l d , C alcluro d - p a n to th e n a te and F yrodoxlne *
A s e p a r a t e s to c k s o l u t i o n o f each o f t h e s e s u b s ta n c e s was p re p a re d by d i s s o l ­
v in g 0 .1 g . i n 100 m l. o f w a t e r .
F o lio A c id t
C o n c e n tr a tio n o f t h i s v ita m in was 2 u g /m l. d is s o lv e d i n
50% e t h a n o l .
R lb o f la v ln e i
One m l. o f g l a c i a l a c e t i c a c id was added t o 25 mg. o f
r i b o f l a v i n e and th e s o l u t i o n d i l u t e d t o 1000 m l. w ith w a te r s o l u t i o n c o n t a in ­
ed 26 u g . p e r m l.
A d en in e, g u a n in e and u r a o l l :
k s i n g l e s to c k s o l u t i o n o f th e s e t h r e e
s u b s ta n c e s was p re p a re d c o n t a in in g I mg. o f e a c h p e r m l.
S o lu ti o n was b ro u g h t
a b o u t by h e a tin g w ith a few c r o p s o f c o n c e n tr a te d h y d r o c h lo r ic a c i d .
X a n th in e i The c o n c e n tr a ti o n o f t h i s s o l u t i o n was a l s o I mg. p e r m l.
was
S o l u t i o n / e f f e c t e d by a few d ro p s o f s t r o n g am monia.
The v a r io u s s to c k s o l u t i o n s o f v ita m in s w ith th e e x c e p tio n o f b i o t i n
w ere renewed a t w eek ly i n t e r v a l s , r i b o f l a v i n and p y ro d o x in e w ere p re p a re d
f r e s h e a c h tim e and th e a d e n in e , g u a n in e , u r a c i l and x a n th in e s o lu ti o n s
w ere renew ed a t tw o-w eek i n t e r v a l s .
A ll s to c k s o l u t i o n s w ere p re s e rv e d u n d e r
to lu e n e in a r e f r i g e r a t o r .
In o rg a n ic S a l t S o lu ti o n At
T h is was composed o f 25 g . o f KgHPO* and
26 g . o f KBgPO* i n 260 m l. o f w a t e r .
In o rg a n ic S a l t S o lu ti o n Bi
7HgO, 0 .6 g . o f
T his s o l u t i o n c o n ta in e d 10 g . o f MgSO4 •
IJSO4 ^ H 2 O and 0 .1 g . o f FeC lg i n 260 m l. o f w a t e r .
Addi­
t i o n o f a few d ro p s o f c o n c e n tr a te d h y d r o c h lo r ic a c id was n e c e s s a r y t o p r e ­
v e n t th e fo rm a tio n o f any p r e c i p i t a t e i n th e m ix tu r e .
*
•
39
—
P r e p a r a t io n o f In o o u lu m i
The lnooulum was p re p a re d by m aking a t r a n s f e r from th e a& ar s ta b
c u l t u r e i n t o a b a o to - p e p to n e , b a o t o - t r ^ t o r e o r o t h (c o m p o s itio n o f t h i s s p e c ­
i a l m edia i s g iv e n on Page 44.1 T h is was in c u b a te d f o r 13-20 h o u rs a t 37°C .
I t was th e n c e n t r if u g e d a e e p t i o a l l y a n d , a f t e r th e s u p e r n a ta n t li q u o r had
b een poured o f f and r e p la c e d by 10 m l. o f 0,9% s t e r i l e s a l i n e s o l u t i o n , i t
was s t i r r e d * i t h a p la tin u m w ire and c e n t r if u g e d a g a i n .
T h is w ash in g o p e ra ­
t i o n was re p e a te d a g a in w ith a n o th e r 10 m l. o f s a l i n e s o l u t i o n .
A f te r th e
t h i r d c e n t r i f u g i n g and re p la c e m e n t w ith 10 m l, s a l i n e s o l u t i o n , 2-5 m l. o f
t h i s s u s p e n s io n was d i l u t e d t o 100 m l. o f s a l i n e s o l u t i o n , th o ro u g h ly mixed
t o in s u r e a u n ifo rm s u s p e n s io n and t h i s d i l u t e inoculum was u sed f o r su b se ­
quent in o c u la tio n .
The f e r m e n ta tio n s w ere c a r r i e d o u t i n p y rex t e s t tu b e s w hich w ere
c a l i b r a t e d t o th e 10 m l. mark*
Aluminum c a p s 2 .5 x 5 cm. w h ich f i t lo o s e ly
o v e r t h e m outh o f th e tu b e w ere u sed in s te a d o f c o t t o n p l u g s .
The aluminum
c a p s veie e a s y t o wash and th e y saved a g r e a t d e a l o f tim e w h ich would o th e r ­
w ise have ueen s p e n t r o l l i n g p lu g s .
A ssay P ro c e d u re i
The fo llo w in g am ounts o f amino a c i d s , v ita m in s u p p le m e n ts , p u rin e and
p y rim id in e b a s e s and m in e r a l s a lt s ,w h e n made up t o a volume o f 600 m l ., g iv e s
s u f f i c i e n t b a s a l medium f o r 100 t u b e s , 6 m l. m ed ia p e r t u b e .
Amino Ac id s
V ita m in S up p lem en ts e t c .
I (+ ) g lu ta m ic a o id
400 mg.
A n eu rin e ( s to c k s o l . d l l . I i 10)
d l a s p a r tic a c id
800 mg.
C alcium d - p a n to th e n a te ( d l l . I t IO)
2 .0 m l.
I (+ ) ly s in e HCl
200
F y rid o x ln e s o l u t i o n ( d i l . 1 :1 0 )
2*0 fill*
(C o n t'd page 40)
mg.
2 .0 m l.
—
40
—
(C o n t'd fpoffl nage 3 9 .)
Ksdno A cids
V ita m in S upplem ents rvTtc.
d l th r e o n in e
200 mg.
R ib o fla v iM s o l u t i o n
d l v a l in e
200 mg.
B lo o t in io a c id s o l u t i o n ( d i l . 1 ,1 0 )
d l i s o le u e in e
200 mg.
p-am ino b e n z o ic a c id s o l o . ( d i l . 1 :1 0 ) 1 .0 m l.
dI
200 mg.
B io t in s o l u t i o n
I ( - ) le u c in e
100 mg.
A denine + g u an in e + u r a c i l s o l u t i o n
1 0 .0 m l.
d l m e th io n in e
I(X) mg.
X a n th in e s o l u t i o n
1 0 .0 m l.
d l p h e n y la la n in e
100 mg.
G lucose
2 0 .0 g .
I (♦ ) a r g i n i n e HCl 50 mg.
Sodium a c e t a t e SEgO
3 5 .2 g .
I (+ ) h i s t i d i n e HC150 mg.
Sodium c h l o r i d e
6 .0 g .
a l a n in e
18 *0 ml 9
^ • 0 sil •
5 .0 m l.
I (-) ty ro s in e
40 mg.
Ammonium s u lp h a te
3 .0 g .
d l try p to p h a n
80 mg.
In o r g a n ic S a l t S o lu ti o n A
5 .0 m l.
I n o r g a n ic S a l t S o lu tio n B
6 .0 m l.
T h is mediumreooBurended by P a rto n -W rig h t (8 ) was used f o r a number o f a s s a y s
and fo u rd t o b e in a d e q u a te in s e v e r a l r e s p e c t s .
U sing t h i s b a s a l medium
la r g e b la n k s w ere o b ta in e d and t h e g row th re s p o n s e was n o t p r o p o r ti o n a l t o
th e amount o f c y s ti n e a d d e d .
T h is was found t o be due p a r t l y t o th e im p u r ity
o f one o f th e am ino a c id s u s e d .
The I (+) g lu ta m ic a c id f i r s t used was
fo u n d by a c o l o r i m e t r i c d e te r m in a tio n t o c o n t a in 0.11% c y s t i n e w hich was r e s ­
p o n s ib le f o r th e h ig h b l a n k s .
Sven b e fo re t h i s d i f f i c u l t y was d is c o v e re d
i t was found t h a t t h e r e w ere s e v e r a l l i m i t i n g f a c t o r s o th e r th a n c y s t i n e .
The v ita m in c o n c e n tr a ti o n was found t o be to o low and th e a d d i t i o n o f s e v e r a l
o t h e r am ino a c id s w ere found t o be s tim u la t o r y t o th e g ro w th o f l - a r a b i n o s u s .
The d i f f i c u l t y o f h ig h b !sinks due t o im p u rity o f th e ! ( + ) g lu ta m ie a c id was
-
41
c o r r e c t e d by u s in g I (+•) g lu ta m ic a c i d p re p a re d by Merck w h ich c o n ta in e d
no a p p r e c ia b le am ount o f c y s t i n e .
A f te r num erous a s s a y s th e fo llo w in g medium
was s e l e c t e d .
For 500 m l. B a s a l Medium
V ita m in s and S u p p le m e n ts , g t o .
Amino A cids
I (+ ) g lu ta m ic a c id
400 mg.
Thiam ine
3 m l.
d l a s p a r t i c a c id
300 mg.
C alcium d - p a n to th e n a te
3 nil *
I (+) L ysine KCl
200 sag.
P y ro d o x in e
3 m l.
d l th r e o n in e
200 mg.
R ib o fla v in e
50 m l.
d l v a l in e
200 mg.
N ic o t in ic a c id
6 m l.
d l i s o le u c in e
200 mg.
F-am ino b e n s o ic a c id
2 m l.
dl
200 mg.
B io t in
I ( - ) lo u c in e
100 mg.
P o lio Acid
d l m e th io n in e
100 mg.
A denine + g u an in e + u r a c i l
15 m l.
d l p h e n y la la n in e
100 mg.
X an th in e
16 m l.
I (+ ) a r g in i n e KCl
50 mg.
G lucose
20. 0 g
I (+ ) h i s t i d i n e KCl
50 mg.
Sodium a c e t a t e SEgO
8 3 .2 g .
I ( - ) ty ro s in e
40 mg.
Sodium c h l o r i d e
5 .0 g .
d l try p to p h a n e
100 mg.
I n o rg a n ic S a l t S o lu tio n A
5 m l.
I ( - ) p r o li n e
60 mg.
I n o rg a n ic S a l t S o lu ti o n B
6 m l.
a la n in e
N ote I
25 m l.
2 m l.
The v ita m in s o l u t i o n s w ere used i n
d l s e rin e
100 rag.
d l n o r le u c in e
100 E g .
th e same c o n c e n tr a ti o n a s th e s to o k
d l n e r v a l ic e
100 tog.
s o lu tio n (n o t d i l u t e ! I tl O ) .
g ly c in e
IOO tog.
42
-
I h s amino a c id s were w eighed o u t and d is s o lv e d i n 100 m l. o f w a te r .
A d d itio n o f two to th r e e d ro p s o f c o n c e n tr a te d h y esro ch lo rio a e id and g e n tle
warm ing waa fo u n d n e c e s s a r y t o b r in g a b o u t co m p lete s o l u t i o n .
T ry p to p h an e
was d is s o lv e d s e p a r a t e l y by warm ing i n th e p re s e n c e o f a few d ro p s o f con­
c e n t r a t e d h y d r o c h lo r ic a c i d , ty r o s i n e by w arm ing w ith .1 B BaCB s o l u t i o n .
The re m a in in g com ponents w are th e n added to ILd amino a c id m ix tu r e and th e
pH a d ju s te d to 6 .8 w ith a few d ro p s o f c o n c e n tr a te d BaCB s o l u t i o n .
was made up t o a volum e o f 500 ml w ith w a t e r .
T h is gave a m ix tu re h av in g
tw ic e th e c o n c e n tr a ti o n o f t h e f i n a l a s s a y medium.
was t r a n s f e r r e d t o eac h t u b e .
and th e s ta n d a r d c y s ti n e c u r v e .
The whole
F iv e m l. o f th e m ix tu re
T h ir ty - o n e tu o e s w ere r e t a i n e d f o r th e b la n k s
The tu b e s w ere s e t up in t r i p l i c a t e and
t o them added s e r i a l l y 6 m l. w a te r ( - b l a n k ) , 0 . 5 , 1 .0 , 1 .5 , 2 . 0 , 2 . 5 , 3 . 0 ,
3 . 6 , 4 . 0 , 5 . 0 , m l. o f s ta n d a r d c y s ti n e s o l u t i o n c o n ta in in g 10 u g . o f c y s ti n e
p e r m l.
To ea c h tu b e s u f f i c i e n t d i s t i l l e d w a te r was added t o b r in g th e
f i n a l volume t o 10 m l.
h y d ro ly s a te s .
The re m a in in g tu b e s w ere used f o r th e a s s a y o f th e
I t was found d e s i r a b l e to c a r r y o u t e a c h a s s a y a t f o u r le v e ls
o f c o n c e n t r a t i o n and i t was i. o a t c o n v e n ie n t t o do t h i s by a d d in g I , 2 , 3 o r
4 m l. o f th e p r o t e i n h y d r o ly s a te t o 5 m l. o f b a s a l medium, th e n b r in g in g th e
s o l u t i o n t o a f i n a l volume o f 10 m l. w ith d i s t i l l e d w a te r .
A f te r a l l s o lu ­
t i o n s had been added th e tu b e s were sh a k e n u n t i l th o r o u g h ly mixed and a u to ­
c la v e d a t 15 l b s . p r e s s u r e , 1 1 8 .6 ° C. f o r t e n m in u te s .
A h ig h e r te m p e ra tu re
o r lo n g e r p e r io d o f a u t o c la v i n g cau sed o a r m e lis a t io n o f th e m edia w hich made
i t w o r th le s s f o r m i c r o b io l o g ic a l a s s a y i n g .
a t e a c h l e v e l o f c o n c e n tr a ti o n
The tu b e s w ere ru n i n t r i p l i c a t e
—
43
—
A l t e r a u t o c la v i n g th e tu b e s w ere allo w e d t o c o o l th e n in o c u la te d u s in g
a 6 c o . s y r in g e (w hich had p r e v io u s ly been s t e r i l i s e d by a u to c la v in g a t 120°c
f o r SO m i n u te s ) .
Two d ro p s o f in o cu lu m were t r a n s f e r r e d t o e a c h t u b e .
The
c o n c e n tr a ti o n o f th e inoculum was found to be a v e r y im p o rta n t f a c t o r , i f th e
inoculum was to o c o n c e n tr a te d o r i f t h e r e was a clum ping o f th e o rg a n is m s,
e r r a t i c grow th c u rv e s r e s u l t e d .
The tu b e s were th e n p la c e d i n a n in c u b a to r f o r 5 6 -4 0 h o u rs a t 37°C .
The te m p e ra tu r e a s w e ll a s th e le n g th o f t h e in c u b a tio n p e r io d w ere found
t o have a v e ry d e f i n i t e e f f e c t on th e s ta n d a r d cu rv e o b ta in e d .
B a rto n -
W right a s w e ll a s s e v e r a l o t h e r i n v e s t i g a t o r s have recommended 3 2 ° C. f o r
72 h o u rs w hich p roved t o be u s a t i s f a c t o r y .
I t m,? fcu iid t h a t 57° C. f o r
3 6 -4 0 h o u rs gave a s ta n d a r d g ro w th cu rv e w h ich was much more r e l i a b l e and had
a s te e p e r s lo p e .
I t was n e c e s s a r y t o s to p g ro w th a f t e r a b o u t 40 h o u rs i n
o r d e r t o s to p th e l a c t i c a c id d ev elo p m en t w h ich ex ceed ed th e b u f f e r in g cap a­
c i t y o f th e Ciediunwhen lo n g e r in c u b a tio n p e r io d s w ere u s e d .
A f te r in c u b a tio n th e c o n te n ts o f th e tu b e s were q u a n t i t a t i v e l y t r a n s ­
f e r r e d t o 126 ml* f l a s k s and l a c t i c a c id formed by th e f e r m e n ta tio n was
t i t r a t e d w ith 0 .1 S NaOH u s in g brcan th y m o l b lu e a s th e i n d i c a t o r .
A s ta n ­
d a rd cu rv e was th e n draw n by p l o t t i n g u g . o f c y s ti n e v s . c e . o f 0 .1 N NaOH.
G rass l e a f p r o t e i n s , v ita m in t e s t c a s e i n , and f r e s h l y p re p a re d
H am m erstein c a s e i n were a s s a y e d by t h i s m eth o d .
C y s tin e was added in known
am ounts b o th b e f o r e and a f t e r h y d r o ly s is t o d e te rm in e th e r e c o v e r y .
Ie u c o n o s to c m e s o n te r lo id e s P-60 was a l s o u sed i n a s s a y in g f o r c y s t i n e .
The method u sed and th e b a s a l m edia f o r L euconostoc m e s e n te r lo ld e s a r e
i n th e fo llo w in g s e c t i o n . The M ic r o b io lo g io a l A ssay o f M e th io n in e .
iv e n
A s ta n -
44
d a rd cu rv e f o r c y s t i n e u s in g J e u c otiosto e in e a e n te rio id e s i s g iv e n on page 60,
F ig u re 6 .
'i he l i v e r - t r y p t o n e - a g a r and b r o t h msdiun used to c a r r y s to c k o u l -
t u r e e was p re p a re d a c c o rd in g t o d i r e c t i o n s g iv e n by Sy$nan and G o rtn e r (S3)
and c o n ta in e d th e fo llo w in g *
L iv e r tr y p t o n e - a g a r (600 m l .)
L iv e r - tr y p to n e - B r o t h ( £ 0 ) m l.)
T ry p to c e
5 .0 grams
T ry p to n e
6 . 0 grama
g lu c o s e
5 .0 grams
G lucose
1 .0 gram
K2HFO4
1 .0 grams
K2HFO4
2 .6 grams
CaCO3
1 .6 grams
T eaet e x tra c t
1 .0 gram
A gar
9 .0 gram s
IA ver e x t r a c t
liv e r e x tra c t
5 0 .0 m l.
6 0 .0 m l.
in o r g a n ic s q l t s A
3 .0 m l.
in o r g a n ic s a l t s E
3 .0 m l.
pH was a d ju s te d t o 7 and 10 m l. d is p e n s e d in e a c h t u b e .
Ie u c o n o s to e rresen -
t e r i o i d e s was c a r r i e d on th e same medium e x c e p t an a g a r s l a n t was used r a t h e r
th a n s ta b c u l t u r e ,.
A n o th er isiedim w hich was oven more e f f e c t i v e in s t im u la t in g
grow th had th e f o llo w in g c o m p o s itio n !
P er IA te r Media
F o r S ta o s
2 .S g .
E ao to p ep to n e
K
y e a s t e x tra c t
2 .6 g .
B aoto tr y p to n o
1%
g lu c o se
1 .0 g .
B acto y e a s t
1.6%
agar
1 0 .0 £ .
Sodium a c e t a t e
1 0 .0 g .
G lucose
6 .0 m l.
I n o r g a n ic s a l t s o l u t i o n A
6 . 0 m l.
**
2 .0 mg.
R ib o f la v in
#
W
B
—
45
—
pH a d ju s te d t o 7 .
F o r th e p r e p a r a t i o n o f H a m e rs te ia c a s e i n th e m ethod g iv e n by M itc h e ll
(5 2 ) was used*
A two q u a r t sam ple o f skimmed m ilk was a d ju s te d t o a pH o f
4 .6 5 u s in g 0 .0 5 K HCI , s t i r r i n g r a p i d l y w i t h a m e c h a n ic a l s t i r r e r .
The r e ­
s u l t i n g p r e c i p i t a t e was washed r e p e a te d ly w ith d i s t i l l e d w a te r by d e c a n ta t io n
and th e n d is s o lv e d by a d d in g s u f f i c i e n t 0 . 1 B HaOH t o b r i n g th e m ix tu re t o a
pH o f 6 . 5 .
The m ix tu re was f i l t e r e d th r o u g h a s u c ti o n f i l t e r u s in g f i l t e r
c l o t h and th e p r e c i p i t a t e d i s c a r d e d .
The c a s e i n was r e p r e c i p i t a t e d u s in g
0 .5 N KCl a t a pH o f 4 . 6 5 , f i l t e r e d , w ash ed , r e d is s o lv e d and p r e c i p i t a t e d
a g a in .
The p r e c i p i t a t e was washed f r e e o f c h l o r i d e s w ith d i s t i l l e d w a te r ,
th e n d e h y d ra te d by a l c o h o l and e t h e r .
The r e s u l t i n g p ro d u c t was w h ite in
c o l o r and a p p e a re d t o have g r e a t e r p u r i t y t h a n th e c o m m e rc ia lly produced
v ita m in t e s t c a se in w h ich had b een used e a r l i e r .
M ic r o b io lo g ic a l D e te rm in a tio n o f M eth io n in e U sin g La u p o n o sto e m e s e n te r io id e s
P -6 0 .
M ic r o b io lo g ic a l m ethod u sed f o r m e th io n in e was t h a t g iv e n by B a rto n W rig h t (8 ) u s in g L eu conostoc m e s e n te r io id e s P -6 0 .
V ery l i t t l e m o d if ic a tio n
o f h is m ethod was fou n d n e c e s s a r y and t h e te c h n iq u e s used w ere th e same a s
th o s e d e s c r ib e d f o r c y s t i n e .
The l a c t i c a c id d e v elo p ed by t h e f e r m e n ta tio n
was t i t r a t e d a g a i n s t 0 .1 N HaOE u s in g brom th y m o l b lu e i n d i c a t o r .
Few d i f ­
f i c u l t i e s w ere e n c o u n te re d and e x c e p t f o r s l i g h t c h a n g e s , th e b a s a l medium
s u g g e s te d by Bar to n - % r ig h t was u s e d .
A ll s to c k s o l u t i o n s w ere p re p a re d a s
b e fo re .
A ssay P ro c e d u r e :
The fo llo w in g am ounts o f am ino a c i d s , v ita m in s u p p le m e n ts ,
e t c . gave s u f f i c i e n t m edia f o r 100 t u b e s .
—
46
Volume Kade Up t o 500 m l.
Amino A cids
d l-
O th e r Components
-A la n in e
1000 mg.
I (+ ) A rg in in e HCl
G lucose
2 0 .0 g .
250 mg.
Sodium a c e t a t e s SfigO
1 9 .9 g .
d l A s p a r tic a c id
800 mg.
Ammonium c h l o r i d e
6 .0 g .
I ( - ) c y s tin e
100 rag.
Sodium c h l o r i d e
5 .0 g .
I (+ ) g lu ta m ic a c id
600 mg.
A denine + g u an in e + u r a c i l s o l o . 1 5 .0 m l.
g ly c in e
100 mg.
X a n th in e s o l u t i o n
1 6 .0 m l.
I (+) h i s t i d i n e HCl
100 mg.
A neurine s o l u t i o n
5 .0 m l.
d I - I s o le u c in e
200 mg.
P y rid o x in s s o l u t i o n
3 *0 sil •
I ( - ) le u c in e
100 mg.
C a le iu m -d -p a n to th e n a te s o l u t i o n
3*0 ml#
I (+ ) ly s in e HCl
125 mg.
B i c o t i n i c a c id s o l u t i o n
6#0 ml#
H o rle u o in e
100 mg.
B io tin s o lu tio n
K orva lin e
100 mg.
F o lic a c id s o l u t i o n
6 .0 m l.
d l JFheity'l a la n in e
100 mg.
p-am ino b e n z o ic a c id s o l u t i o n
2 .0 m l.
I ( - ) p ro lin e
100 mg.
^ ib o f la v i n e
3 .0 mg.
d l s e rin e
100 sig.
I n o r g a n ic s a l t s o l u t i o n A
5 .0 m l.
d l th r e o n in e
500 mg.
I n o r g a n ic s a l t s o l u t i o n B
5 .0 m l.
d l tr y p to p h a n
100 mg.
I ( - ) ty ro s in e
100 mg.
d l v a lin e
100 mg.
5 0 .0 m l.
The am ino a c id s w ere w eighed o u t and d is s o lv e d in 100 m l. o f w a te r .
th e re m a in in g com ponents o f th e m edia w ere a d d e d , th e pH a d j u s t e d to 6 .8 w ith
sodium h y d ro x id e th e whole made up t o 600 ml* w ith w a t e r .
tw ic e th e c o n c e n tr a ti o n o f th e f i n a l a s s a y medium .
to each tu c e .
T h is s o l u t i o n had
F iv e m l. w ere t r a n s f e r r e d
-
47
-
Xhe s ta n d a r d m e th io n in e s o l u t i o n c o n ta in e d 10 u g . p e r m l.
F o r th e s e t
up o f an a s s a y th e n e c e s s a r y number o f tu b e s w ere r e t a i n e d f o r b la n k s and th e
d e t e r m in a tio n o f th e s ta n d a r d m e th io n in e c u r v e .
As i n c y s t i n e th e t o t a l
volume o f e a c h tu b e was made up t o 10 m l. w ith w a te r .
Each a s s a y was c a r r i e d o u t a t f o u r l e v e l s o f c o n c e n t r a t i o n .
The h y d ro ­
l y s a t e s w ere d i l u t e d t o c o n t a in an a p p r o p r ia te amount o f m e th io n in e .
The
c o n c e n tr a ti o n l e v e l s t h a t w ere u sed w ere 1 .0 , 2 . 0 , 5 . 0 , and 4 . 0 m l. o f hy­
d r o ly s a t e *
A f t e r t h e a d d i t i o n o f th e s ta n d a r d am ino a c id s o l u t i o n o r h y d r o ly s a te s
and th e a d ju s tm e n t o f th e volum e o f th e tu b e s to 10 m l ., th e tu b e s w ere
co v ered w ith alum inum cap s and s t e r i l i z e d by a u t o c la v in g f o r 10 m in u te s a t
15 pounds p r e s s u r e ,
1 1 8 .8 ° C .
The tu b e s th e n allo w ed t o e o o l and in o c u la ­
te d w ith le u c o n o a to c .T e s e n te r io id e s .
The same p ro c e d u re was weed i n p r e p a r ­
in g th e in ocu lu m a s in th e c y s ti n e d e t e r m i n a t i o n .
c u b a te d a t 57° Ce f o r 5 6 -4 0 h o u r s .
The tu b e s w ere th e n i n ­
The l a c t i c a c id fo rc e d was t i t r a t e d w ith
0 .1 B SaOH u s in g brom th y m o l b lu e i n d i c a t o r .
L ea f p r o t e i n p r e p a r a t i o n s , v ita m in t e s t c a s e i n , and f r e s h l y p re p a re d
H am rsfcein c a s e i n w ere a s s a y e d by t h i s m ethod*
A s ta n d a r d m a th io n ln e c u rv e
u s i n t, L euconostoc m a s e n te r lo ld e s P^60 i s g iv e n on Page ^ q, F ig u re 7*
♦
48
TABUS NO. I
F tosulte o f !■ ' i .'I c x o ld - S tham ol E x t r a c t i o n o f B o u teIo u a g r a c i l l i s Cut 8 -2 3 -4 6
H eig h t o f f r e s h g r a s s a n a ly s e d . . . . . . . . . . . . . . . . . . . . . . .
P e r c e n t m o is tu re on a i r d ry b a s i s . . . . ................... . . . . . . . . . . . . . . . . . .
P e r c e n t n i t r o g e n in a i r d ry sa m p le .
w eig h t lo s s by a c e to n e e x t r a c t i o n o f f r e s h g r a s s
. . . . . . . . . . . . . . . . . . . .
P er c e n t n i t r o g e n o f g r a s s a f t e r a c e to n e e x t r a c t i o n . . . . . . . . . . . . . . . . . . .
Vieight o f r e s id u e a f t e r f o m l c a c id e x t r a c t i o n ............................
P e r c e n t n i t r o g e n i n r e s id u e a f t e r fo rm ic a c id and a c e to n e e x t r a c t i o n . .....................................
P e r c e n t n i t r o g e n i n c a rb o h y d ra te r e e id u e
P er c e n t n i t r o g e n i n fo rm ic a c id s o lu b le f r a c t i o n . . . . . . . . . . . . . . . . . . . .
P er c e n t n i t r o g e n i n p r o t e i n e x t r a c t • .................................. . . . . . . . . . . . . . . . . .
1196.0 g .
3 8 .1 %
1.31#
4 2 0 .1 g .
1.39%
30 6 .7 g .
0.51%
0.56%
2.56%
4.04%
I
OO
Summary o f N itro g e n Bl s t r i b u t i o n A ite r E x tr a c tio n
W eight o f f r e s h g r a s s a n a ly s e d . . . . . . . . . . . . . .
W eight o f n it r o g e n i n sample . . . . . . . . . . . . . . .
P e r c e n t p r o t e i n found (% H x 6 . 2 5 ) . . . . . . . . . . . .
N itro g e n e x t r a c t e d grams . . . . . . . . . . . . . . . . .
p e r c e n t e x tra c te d
.
.
Form ic a c id s o lu b le n it r o g e n grams .
p e r c e n t o f to t a l n itro g e n . ,
N itro g e n i n a c e to n e s o lu b le f r a c t i o n
p e r c e n t o f t o t a l n i t r o g e n . • ...................
. . . . .
N itro g e n i n c a rb o h y d ra te r e s id u e . . .
. . . . . . . . . .
per c e n t of t o t a l n itro g e n .
N itro g e n i n r e s id u e a f t e r fo rm ic a c id + a c e to n e e x t r a c t i o n
per ce n t o f t e t a l n itro g e n . . . . . . . . . . . . . .
...................
. . . . .
. . . . .
. . . . .
. . . .
. . . .
. . . .
. . . .
. . . .
. . . .
. . . .
. . . .
.
.
.
.
.
.
.
.
................................ ....
. . . . . . .
. . . . . .
. . . . . . . . . . . . . . .
................... . . . . . . . . . .
. . . . . . . . . . . . . . .
1196.0 g .
9 .6 9 g .
8 .1 9 %
1.74 g .
18%
6 .8 0 g .
60%
,3 0 g .
3 .1 %
.16 g ,
1,7 %
1.66 g .
16 %
\
TABLS KO. I I
R e s u lts o f Sodium D odeoyl S u l f a t e E x t r a c ti o n
B o u te I o u a g r a c i l l i s ( o u t 8 - 2 9 - 4 6 )
M i g h t o f a i r d r y s a m p l e ...............................................................................................................................................
P er c e n t n i t r o g e n i n s a m p le . ................................................................................
W eigh t o f r e s id u e a f t e r e x t r a c t i o n ......................................................................
P er c e n t n i t r o g e n i n r e s i d u e .............................................................. . . . ..............................................
P e r c e n t n i t r o g e n i n p u r i f i e d p r o t e i n p r e p a r a t io n
............................... . . . . . . . . .
N it r o g e n D i s t r i b u t i o n
«
A fte r
2 0 0.00 g .
1.42 fa
1 5 7 .9 6 g .
0 .6 3 %
8 .0 4 %
e x tr a c tio n
W eig h t o f a i r d r y s a m p l e .......................... ................................................................................................. . . . '
P er c e n t p r o t e i n fo u n d OS N x 6 . 2 6 ) . • • • •
..................... . . . . . . . . . . . . . .
T o t a l w e ig h t o f n i t r o g e n i n sam p le • • .................................... . . . . . . . . . . . . . .
.T eigh t o f n i t r o g e n e x t r a c t e d .......................... ........................................ ...................................
p e r c e n t o f t o t e I n it r o g e n
W eigh t o f n i t r o g e n i n r e s id u e a f t e r e x t r a c t i o n
. . .
p er c e n t o f t o t a l n it r o g e n
..........................................................................................................................
.....4 . ... .... ........
. . . . . . . . . . ..........
2 0 0 .0 0 g .
8 .8 9 %
2 .8 4 g ,
.7 1 g .
26.
%
g.
,9 9
35.
%
The F o llo w in g G ra sse s w ere S u b je c te d t o Sodium D odeoyl S u lf a te E x t r a c ti o n
G rass E x tr a c te d
A gropyron s m i t h ! ! ( c u t 5 - 1 0 - 4 6 )
A grop yron s m i t h ! ! ( c u t 7 - 1 5 - 4 6 )
Wt. o f
A ir Dry
Sam ple
5 0 .0 g .
5 0 .0 g ,
in
Sam ple
3 .9 9 %
1 .5 4 %
Wt. H
in
Sam ple
1 .9 9 g .
.7 7 g .
A g ro p y ro n c r is t a t u m
A gro p yron c r i s t a t u m
5 0 .0 g .
5 0 .0 g .
4 .3 4 %
1 .1 9 %
2 .1 7 g .
.6 0 g .
(c u t 6 -1 0 -4 6 )
(c u t 7 -1 5 -4 6 )
% N
W t. N
% o f T o ta l
i n Pro
E x t r a o te d i n P u r i- t e i n pr e fled Protein p a r a t io n
.8 5 5 . ' 2 . 7 %
1 2 .5 3 %
•2 9 g .
3 7 .6 %
1 1 .2 3 %
.8 2 g .
.2 3 g .
3 7 .6 %
3 7 .6 %
1 2 .7 1 %
1 1 .5 1 %
1
£
50
-
TAJBLS SO. I l l
Record o f H y d ro ly s is
P r o t e i n S a m p le
G ram s/lO O c c . I iy d r o ly z a t e
D ate
H y d r o ly z e d
So. o f
H y d ro ly z a te
B o u t e lo u a g r & o i l l i s
(o u t 8 -2 3 -4 6 )
I g ./ i o o c e .
1 1 -2 6 - 4 6
8A + B
B o u t e lo u a g r a o l l l i e
(o u t 8 -2 3 -4 6 )
I g ./ i o o c c ,
1 -2 1 -4 7
GA + B
A gro p y ro n s m .i t h i i
( o u t 5 - 1 0 —4 6 )
I g ./ i o o CO.
1 -2 1 -4 7
I A + B
A g ro p y ro n s m i t h ! !
( c u t 5 —ID—4 6 )
I g ./lO O CO.
3 -2 -4 7
21
A gropyron s m it h ! I
(c u t 7 -1 5 -4 6 )
I g ./ i o o c e .
1 -2 1 -4 7
2A + B
A g ro p y ro n s m i t h I !
(o u t 7 -1 5 -4 6 )
I g ./io o c c .
3 -2 -4 7
22
A g ro p y ro n c r ie t a t u m
(o u t 6 -1 0 -4 6 )
I g ,/ i o o CO.
1 -2 1 -4 7
BA * B
A gro p y ro n c r is t a t u m
( o u t 6 - 1 0 —4 6 )
I g ./ i o o c e .
3 -2 -4 7
23
A gro p y ro n c r is t a t u m
(c u t 7 -1 5 -4 6 )
I g ./lO O CO.
1 -2 1 -4 7
4A + B
A g ro p y ro n c r is t a t u m
(c u t 7 -1 5 -4 6 )
I g ./ i o o c c .
3 -2 -4 7
24
V ita m in T e s t C a s e in
I g ./lO O c c .
1 1 -2 2 -4 6
GA
V ita m in T e s t Case i n
I g ./ i o o CO.
1 -2 1 -4 7
V ita m in T e s t C a s e in
I g ./ i o o c c . + 1 0 m g. c y s t i n e
+ 10 m g. m e th io n in e
1 1 -2 2 -4 6
SB
V ita m in T e s t C a s e in
I g ./ i o o CO.
3 -2 -4 7
25
F r e s h ly p rep a re d
H a m e r s te in C a s e in
I g ./lO O CO.
2 -2 1 -4 7
ISA * B
F r e s h l y p rep a red
H a m e r s te in C a s e in
I g ./ i o o c e .
3 -2 -4 7
26
9A + B
—
V I.
51
—
DISCUSSI OM OF EXPERIMSBTAL RESULTS
D is c u s s io n o f P r o te in E x t r a c t io n ;
The r e s u l t s
o b ta in e d u s in g A . A . A lb a n e s e 1a m ethod ( I ) a r e g iv e n on
T a b le I , p a g e 4 8 .
The p e r c e n t o f t o t a l p r o t e i n e x t r a c t e d was 16$ and th e
p e r c e n t p r o t e i n o f t h e f i n a l p r o d u c t o n ly 2 5 $ .
was n o t s u i t a b l e
I t a p p e a r s t h a t t h i s m ethod
f o r th e e x t r a c t i o n o f g r a s s p r o t e i n s .
A lb a n e s e fo u n d t h a t a p p l i c a t i o n o f t h e fo r m ic a c i d - e t h a n o l method t o
d r i e d s e e d m e a ls or p u l v e r i s e d d e h y d r a te d v e g e t a b l e s was u n s a t i s f a c t o r y
o w in g t o
th e f o r m a t io n o f g e l s and l o s s o f d i s p e r s e d p r o t e i n a c h ie v e d
t h e s e p r e p a r a t io n s .
in
In h i s e x p e r im e n ts 6 0 t o 70$ o f t h e t o t a l n i t r o g e n was
rem oved a f t e r 6 e x t r a c t i o n s w i t h fo r m ic a c i d , and he s t a t e s t h a t th e a d a p ta ­
b ility
o f th e method t o f o o d s t u f f o t h e r th a n f r e s h v e g e t a b l e s i s
th e p a r t ic le
s ite
and p h y s i c a l c h a r a c t e r i s t i c s
l i m i t e d by
o f th e p r o d u c t.
The fo r m ic a c i d t r e a tm e n t b r o u g h t a b o u t a c e r t a i n am ount o f h y d r o l y s i s
o f t h e p l a n t p r o t e i n ano c a r b o h y d r a te m a t e r i a l w h ic h r e s u l t e d i n c o n s i d e r ­
a b le hum in f o r m a t io n .
Due t o th e h i g h l i g n i n and e a r b o h y d r t e
c o n t e n t o f th e
g r a s s u s e d , a c o l l o i d a l d i s p e r s i o n r e s u l t e d w h ic h d id n o t p r e c i p i t a t e o u t
on a d d i t i o n o f e t h a n o l .
in s o lu b ility
S in c e th e
s e p a r a t io n o f th e p r o t e i n d ep en d s upon th e
o f th e c a r b o h y d r a t e s i n th e f o r a i o a c i d - e t h a n o l m i x t u r e , t h e f a c t
t h a t t h e s e m a t e r i a l s w ere n o t r e n d e r e d i n s o l u b l e by t h e a d d i t i o n o f e t h a n o l
i n d i c a t e s t h a t t h e c a r b o h y d r a t e - p r o t e i n r e l a t i o n s h i p w as s u c h a s n o t t o b e
s e p a r a b le by t h i s t e c h n i q u e .
Sodium d o d e o y l s u l f a t e e x t r a c t i o n p ro v ed t o be v e r y s a t i s f a c t o r y .
r e s u lts
o f s e v e r a l e x t r a c t i o n s a r e g i v e n on T a b le I I , page 4 9 .
The
iso e f f o r t w as
made t o e f f e c t a q u a n t i t a t i v e s e p a r a t i o n , y e t y i e l d s a s h ig h a s 43% w ere ob­
ta in e d .
The p e r c e n t o f t o t a l p r o t e i n e x t r a c t e d v a r ie d som ew hat w it h th e a g e
—
o f th e g r a s s .
52
—
In g e n e r a l / t h e l a t e r th e c u t t i n g th e lo w er th e y i e l d .
T h is
could b e due t o s e v e r a l f a c t o r s , one o f w hich m ight be th e d e c r e a s e in
p r o t e i n c o n te n t and th e in c r e a s e i n l i g n i n and c a rb o h y d ra te m a t e r i a l i n the
more m atu re g r a s s e s , w h ich would make c o m p lete e x t r a c t i o n more d i f f i c u l t .
The f u n c ti o n o f l i g n i n i n th e p l a n t i s a p p a r e n tly t o g iv e s t r e n g t h and r i g i d ­
i t y t o th e c e l l w a l l s .
The u n l i g n i f l e d t i s s u e o f th e y o u n g e r g r a s s e s i s u su ­
a l l y s o f t , c o n ta in s a h ig h e r p e rc e n ta g e o f w a te r and h a s a much low er b re a k ­
in g s t r e n g t h w h ich would te n d t o make e x t r a c t i o n much l e s s d i f f i c u l t .
The
f i n a l p ro d u c ts c o n ta in e d a s h ig h a s 80% p u re p r o t e i n m a t e r i a l w h ich I s co n ­
s i d e r a b l y b e t t e r th a n r e s u l t s o b ta in e d by th e fo rm ic a c i d - e th a n o l e x t r a c t i o n .
The p u r i t y o f th e f i n a l p r e p a r a t io n was lo w er i n th e more m atu re g r a s s e s .
Lugg (2 8 ) used a c e to n e e t h e r , w a te r f o r t h e plasm oly s in g a g e n ts and was a b le
t o e x t r a c t o n ly 27.2% o f th e t o t a l p r o t e i n from fetedic a g o - s a t i v a ( lu c e r n e )
w ith a f i n a l p r o d u c t c o n t a in in g a p p ro x im a te ly 80% p r o t e i n .
42 .8
P er c e n t T r a n s m i s s i o n
t
^ Ti r e in V in u te s
F ig u r e 2
C o n c e n t r a ti o n o f c y s t i n e i n grams
__________
Curve o b ta in e d u s in g f r e s h l y p re p a re d sodium s u l f i t e r e a g e n t .
__________
Curves o b ta in e d when I , 3 , and 7 day old sodium s u l f i t e s o l u t i o n s were
used •
S p e c t r a l T r a n s m i s s i o n Curve f o r c y s t i n e u s in g Lugg' s c o l o r i m e t r i c m ethod.
S p e c tr o p h o to m e te r r e a d in g s t a k e n a t 650 m^i
TABIE NO.
nr
R e s u lts o f C y s tin e D e te rm in a tio n s U sing Lugg1s M o d if ic a tio n o f F o lin -M a re n s l Method
A -c ris ta tu m
A - s m ith ii
P r o te in E x tr a c t
B -g ra c illie
D ate g r a s s was o u t
6 -1 0 -4 6
7 —16—46
6 -1 > 4 6
7 -1 5 -4 6
8 -2 9 -4 6
D y d ro ly s a te number
21
22
25
24
8A
8B
3 -2 0 -4 7
3 -2 0 -4 7
3 -2 0 -4 7
5 -2 0 -4 7
12-29-46
12-30-46
P e r c e n t c y s ti n e r e p o r te d on th e b a s i s o f
16% 8 .
1 .1 1
1.11
1.17
1.17
1 .0 6
1.05
.96
.96
I . DS
1.1DS
U D2
I . 02
0 .7 8
0 .7 8
0 .8 0
0 .8 0
0 .5 0
0 .6 2
0 .6 4
0 .6 0
0 .6 4
0 .6 6
0 .6 6
Average v a lu e %
1 .1 4
1 .0 1
I . 03
0 .7 9
0 .6 7
0 .6 2
D ate o f d e te r m in a tio n
V ita m in T e s t
C a se in
H y d ro ly s a te Number
A verage V alue %
5-A
0 .3 1
5-B
C y stin e added
1 .2 7
3-29-46
F r e s h ly P rep ared H am erste in C asein
ISA
18B
0 .4 7
0.5C
26
0 .6 0
Per C ent C y s tin e t& co v ery ; c y s ti n e added a f t e r h y d r o ly s is o f p r o te in *
E y d ro ly s a te SB - 96% r e c o v e ry ; H y d ro ly s a te 21 - 93% re c o v e ry
P e r C ent C y s tin e R eco v ery ; c y s ti n e adtiet, b e f o r e h y d r o ly s is *
H y d ro ly s a te SB - 37% re c o v e ry
NOTEi
A ll p e rc e n ta g e s o f m e th io n in e and c y s tin e r e p o r te d i n t h i s t h e s i s a re on th e b a s i s o f 16% n it r o g e n .
■
D is c u s s io n o f C y s tin a
56
t e r a i n a t ions ;
Lugg1S m o d i f ic a t io n o f t h e F o lin - K a r e n z i method f o r th e d e te r m in a tio n
o f c y s ti n e p r e s e n ts num erous d i f f i c u l t i e s .
Kolybdenum m ust be c a r e f u l l y
removed from th e phosphot u n g s t i c a c id r e a g e n t t o av o id r e a c t i o n s w ith t y r o ­
s i n e , try p to p h a n and p h e n o lic g ro u p s g e n e r a l l y .
Most h y d r o ly s a te s w hich
have t o be a n a ly z e d f o r c y s t i n e c o n t a i n o th e r s u b s ta n c e s w hich c o n t r i b u t e
t o th e c o l o r a t i o n d e v e lo p e d .
Those s o lu b le p ro d u c ts a r i s i n g from th e d i g e s ­
t i o n o f c a rb o h y d ra te m a t e r i a l w ith m in e r a l a c id w i l l a ls o red u ce th e
r e a g e n t.
There i s re a s o n t o b e l i e v e t h a t any s u f f i c i e n t l y s o lu b le s u lp h -
h y d ry l compound w i l l re d u c e th e r e a g e n t and th e same may be s a i d o f any d i ­
s u lp h id e w hich w i l l r e a c t w ith s u l p h i t e t o form a s u lp h h y d r y l compound.
I t i s c l e a r th e n t h a t th e r e a c t i o n w ith p h o s p h o - 1 8 - tu n g s ti c a c id i s n o t o n ly
u n s p e c if i c f o r c y s t i n e among th e s u lp h lty d ry I com pounds, b u t i t i s f u r t h e r
u n s p e c if i c f o r th e s u lp h h y d r y l compounds th e m s e lv e s .
C o r r e c tio n has been
made f o r th e s e e x tra n e o u s r e d u c e rs by p r e p a r in g an unknown b la n k c o n ta in ­
in g m e rc u ric c h l o r i d e w h ich i n h i b i t s c o l o r developm ent due t o c y s te in e and
th e c y s te i n e c o n te n t i s th e n o b ta in e d by d i f f e r e n c e .
The pH o f th e c o l o r
r e a c t i o n m ust be c a r e f u l l y m a in ta in e d a t a pH o f 5 .7 and th e q u a n t i t y o f
th e r e a g e n ts added m ust be c a r e f u l l y c o n t r o l l e d .
Many o f th e s e d i f f i c u l t i e s
have b een d is c u s s e d by Lugg ( 2 8 ) .
F ig u re I , page 6 3 , i l l u s t r a t e s th e im p o rtan ce o f c o n t r o l l i n g th e le n g th
o f tim e f o r c o l o r d e v e lo p m e n t.
T em p eratu re h as a p p a r e n tly l i t t l e e f f e c t
on th e r a t e o f c o l o r dev elo p m en t and el I r e a c tio n s w ere c a r r i e d o u t a t
room te m p e r a tu r e .
- 57
F ig u r e 2 , page 5 4 , i l l u s t r a t e s th e im p o rta n c e o f u s i n g f r e s h sodium
s u lf ite s o lu tio n .
A change o f s lo p e o f t h e s t a n d a r d c u rv e i s observed
when old s u l f i t e s o l u t i o n i s u s e d , how ever, w ith f r e s h l y p r e p a r e d s o l u t i o n
t h e same s t a n d a r d c u rv e may be r e p e a te d e a c h d a y .
The r e s u l t s o f c y s t i n e d e t e r m i n a t i o n s u s i n g Lugg1s m o d i f i c a t i o n of
t h e F o lin - M a r e n z i method a r e g iv e n on Table No. IV , page 5 5 .
The v a lu e s
o b ta in e d f o r c y s t i n e i n v i t a m i n t e s t c a s e i n a g r e e q u i t e w e l l w i t h t h o s e
g iv e n by B laok ariti B o l l i n g ( 9 ) .
The v a lu e g iv e n b y B la ck and B o l l i n g f o r
t h e p e r c e n ta g e c y s t i n e i n c a s e i n is 0 .3 6 _+ O»04%.
C a l c u l a t e d t o 1 6 .0 p e r
c e n t n i t r o g e n , th e v a l u e shown on Table IV i s 0.31% c y s t i n e c a l c u l a t e d on
th e b a s i s t h a t t h e p u re p r o t e i n c o n t a in e d 16% n i t r o g e n .
10 _ _
F ig u re
6
__
S ta n d a rd Growth Curve f o r c y s t i n e
u s in g I .- a r a c in o s u s 17-5 i l l u s t r a ­
t i n g th e h ig h b la n k v a lu e o b ta in e d
when impure I (+) g lu ta m ic a c id was
C o n c e n t r a ti o n of C y s tin e i n ^g
F ig u re
C o n c e n t r a t i o n o f c y s t i n e i n jig
7
___
6—
O
o
F ig u re
O
4
S ta n d a rd C y s tin e Growth Curve f o r
Leuconostoc me s e n te r o id e s P -6 0 .
Sg
§
o
C o n cen tratio n o f c y s tin e in
9
TABLE KO. V
JS eeu lte o f C y s t in e A ssa y E m p loyin g L a c t o b a c i l l u s - a r a b in o e u s 1 7 -5
P r o te in e x t r a c t
D a te g r a s s w as c u t
H y d r o ly s a te number
[A t e o f a s s a y
A -s m ith li
6 -1 0 -4 6
21
5 -2 6 -4 7
1 .6 2
1 .6 9
1 .7 6
1 .8 8
1 .7 6
IA
4 -1 0 -4 7
Ba
2 -2 6 -4 7
1 .1 9
1 .2 5
1 .1 9
1 .2 6
1 .2 6
1 .0 7
1 .0 6
1 .9 9
1 .9 6
1 .9 6
1 .7 3
1 .6 6
7 -1 5 -4 6
2B
3 -6 -4 7
1 .9 0
1 .9 0
1 .8 4
1 .8 4
1 .9 1
22
5-2 5 -4 7
1 .3 1
1.66
1 .6 6
1 .6 1
1 .6 1
1 .4 9
A -o r is ta tu o
7 -1 5 -4 6
4A
4B
24
3 -6 -4 7
2 -6 -4 7
3 -2 6 -4 7
1 .0 8
1 .2 5
1 .2 8
1 .0 8
1 .3 6
1 .3 5
1 .3 5
1 .1 4
1 .2 6
1 .1 1
1 .6 3
1 .6 0
1 .3 9
1 .1 8
1 .2 9
1 .2 9
1.22
1 .2 6
1 .2 9
A v era g e V a lu e %
P r o te in e x t r a c t
D a te g r a s s w as c u t
h y d r o ly s a t e number
D ate o f A ssay
1 .7 4
1.86
1.88
1 .5 2
1 .1 7
1 .2 9
1 .3 0
A -c rlsta tu n B -g r a c llH s H am ersteln C a a e ln
7 -1 6 -4 6
9 —2 9—46
24
ISA
18B
6-B
4 -1 0 -4 7
3 -6 -4 7
3 -8 -4 7
3 -8 -4 7
1 .3 5
1 .1 1
1 .0 1
A v e ra g e v a lu e %
1 .1 8
1 .1 4
1 .1 6
1.12
.8 9
•76
•8 1
1 .0 6
I • 14
1 .1 8
1.12
.4 7
.3 9
.3 2
.4 7
.3 1
.3 9
.4 7
•5 1
0 .3 9
0 .3 9
N o t e i Per c e n t c y s t i n e re c o v e r y ; c y s t in e added
a f t e r h y d r o ly s is .
yd r o l y s a t e * 2 1 - 101%
recovery.
TABLE RO. VI
rS e e u lts o f C y s tin e A ssa y U eing Leuoonostoc- •m e s e n te rio itie s P-60
P r o te i n E x tr a c t
Date g r a s s was c u t
H y d ro ly z a te number
D ate o f a s s a y
A verage V alue %
A -s a ith li
7 -1 6 -4 6
5 -1 0 -4 6
22
I-A
4 -1 4 -4 7
4 -1 4 -4 7
1 .5 7
.8 4
.85
1.11
1.52
1 .1 7
1.53
1 .2 0
1 .0 7
1 .1 9
1.15
1 .0 1
.94
1.1 1
1.12
£ -1 0 -4 6
S-A
4 -1 4 -4 7
1.42
1.48
1 .2 9
1.2 5
1.35
A -o ris ta tu m
6 -1 0 -4 6
25
4 -1 9 -4 7
7 -1 6 -4 6
24
4 -1 9-47
1 .2 1
1.08
1 .1 1
.9 4
1.33
.8 9
.88
.7 9
.97
.9 0
.7 9
.74
.71
.6 6
.66
1.03
0 .7 8
H a m e rste in
C a se in
18a
4 -1 9 -4 7
.46
.63
.52
.5 9
.6 9
0 .6 4
G y a tln g Assay U sin g ? > a c t o b a c i i l i s - a r a b I posus 1 7 - 5 i
A g r e a t d e a l o f d i f f i c u l t y w as e n c o u n t e r e d i n o b t a i n i n g a s u i t a b l e b a s a l
m e d ia f o r t h i s o r g a n ism and num erous m o d i f i c a t i o n s w ere t r i e d b e f o r e a s u i t ­
a b le medium was f i n a l l y
o b ta in e d ,
t h e m o st d i f f i c u l t
t h e e l i m i n a t i o n o f h ig h b la n k s w h ic h r e s u l t e d
h ig h c y s t i n e v a l u e s .
p ro b le m i n v o lv e d was
i n e r r a t i c s ta n d a r d c u r v e s and
F ig u r e S i l l u s t r a t e s a t y p i c a l s ta n d a r d c u r v e w it h a
h i g h b la n k and sh ow s how t h e t i t r a t i o n s v a r y i n t u b e s c o n t a i n i n g e q u a l c o n c e n ­
tr a tio n s o f c y s t in e .
u n d o u b te d ly d u e t o
I t a p p e a r e d t o b e q u i t e o b v io u s t h a t th e h ig h b la n k w as
th e im p u r it y o f so n # am ino a c id
i n t h e m ediu m .
By t r i a l
and e r r o r e l i m i n a t i o n , i t was fou n d t h a t t h e I (+ ) g Iutam c a c i d b e in g u sed
was r e s p o n s i b l e f o r t h i s d i f f i c u l t y .
When I ( + ) g lu t a m ic a c id
o f a b e tte r
q u a l i t y w a s u sed t h e h ig h b la n k d is a p p e a r e d and a more s t a b l e gro w th c u r v e
w as o b t a i n e d .
bo
A c o l o r i m e t r i c d e t e r m i n a t i o n w as made on th e I (+ ) g lu t a m ic
id w h ic h had b e e n fo u n d im pure and i t w as d is c o v e r e d t h a t t h i s p r o d u c t
c o n t a in e d
1 . 1 m g. o f c y s t i n e p e r g ra m .
T h is r e s u l t e d
o f c y s t i n e p e r a s s a y tu b e and w ou ld e a s i l y
i n an a d d i t i o n a l 5 u g .
a c c o u n t f o r th e h ig h b la n k o b t a i n ­
ed.
I t w as n e c e s s a r y t o
I n c r e a s e t h e v i t a m i n c o n c e n t r a t i o n s t o a p p r o x i­
m a t e ly t e n t i m e s t h e c o n c e n t r a t i o n s g i v e n i n t h e l i t e r a t u r e and t o add o t h e r
g ro w th s t i m u l a t i n g s u b s t a n c e s .
A lth o u g h th e s ta n d a r d c u r v e s w h ic h were
f i n a l l y o b t a in e d show ed some i n c o n s i s t e n c i e s , th e r e s u l t s w e r e c o n s i d e r a b l y
b e t t e r w hen t h e medium g i v e n on p age 4 Iw as u s e d .
s ta n d a r d g ro w th c u r v e u s i n g t h i s
A very
F ig u r e 4 , p age 5 9 , sh ow s a
new m e d ia .
i n t e r e s t i n g o b s e r v a t i o n s h o u ld b e m e n tio n ed i n r e g a r d t o th e
c y s t i n e g ro w th c u r v e s o b t a in e d i n t h i s e x p e r im e n t .
For e v e r y c y s t i n e c u r v e
-
64
o b ta in e d th e r e was a p o in t o f i n f l e c t i o n a t a c y s t i n e c o n c e n tr a ti o n o f
15 u g .
C u rio u s ly enough t h i s o b s e r v a tio n h as n e v e r b e e n r e p o r te d i n th e
lite ra tu re #
A lth o u g h th e r e a s o n f o r t h i s change i n s lo p e i s unknown,
s e v e r a l e x p l a n a ti o n s m ig h t be g iv e n *
The m ost re a s o n a b le e x p l a n a tio n seems
t o be t h a t a f t e r th e c y s ti n e c o n c e n tr a ti o n re a c h e s a c e r t a i n l e v e l , a n o th e r
a c id p ro d u cin g m edian ism g oes i n t o e f f e c t in c r e a s in g th e r a t e o f a c id p ro ­
d u c tio n .
I t i s known t h a t c y s t i n e a c t s a s an enzyme a c t i v a t o r .
C y stin e i s
n e c e s s a r y f o r th e s y n th e s is o f g lu t a t h i o n e { -g lu ta m y 1 -c y s t e i n y I g l y c i n e ) ,
^ u a s t e l (2 1 ) p r e s e n ts e v id e n c e f o r th e c o m b in a tio n of g lu t a t h i o n e w ith m e th y l
g ly o x a l and p ro p o se a h y p o th e s is o f g ly o x a la s e a c t i o n w h ich in c lu d e s th e
IP
IP
fo rm a tio n o f an a d d i t i o n compound composed o f m e th y l g ly o x a l and g l u t a t h i o n e i
■•
CiO
Se
!.:•
^
>» C Sv
H— C : 0
CH(OH).S . R
t e t h y l g ly o x a l + G lu ta th io n e
A d d itio n compound
The a d d i t i o n oompou d i s th e n decom posed w ith th e fo rm a tio n o f l a c t i c a c id
and g lu t a t h i o n e
%
C tO
>
H2 O
*■
CH(OH)
C H (O H ).S.R
CO. OH
A d d itio n compound
L a o tio a c id
+
R. S . H
O lu ta th io n e
I t seems e n t i r e l y p o s s ib le t h a t some a c id p ro d u c in g m echanism su ch a s t h i s
may go i n t o e f f e c t when th e c y s ti n e c o n c e n tr a ti o n re a c h e s a c e r t a i n l e v e l .
I f t h i s assu m p tio n i s t r u e , i t would a c c o u n t f o r th e p o in t o f I n f l e c t i o n
found i n s ta n d a r d c y s ti n e grow th c u r v e s .
Tabto No. V, page 6 1 , shows th e
— 66 •»
r e s u l t s o b ta in e d b y a s s a y i n g c y s t i n e w ith L - a r a t ln o s u s
1 7 -6 .
The v a l u e s
a r e i n a l l c a s e s h ig h e r th a n t h o s e o b t a in e d b y o th e r m eth od s and u n t i l a
more th o r o u g h s tu d y h a s b e e n made o f t h e c y s t i n e m e ta b o lis m o f L -a r a b ln o s u s
n o t much v a l u e c a n be a t t a c h e d t o t h e s e r e s u l t s .
C y s t in e D e t e r m in a t io n U s in g L e u c o n o sto c R e s e n t e r i o i d e s P - 6 0 :
The same b a s a l medium w as u se d f o r t h e c y s t i n e a s s a y a s t h e m e th io n i o i n e a s s a y e m p lo y in g L e u c o n o sto o m s s e n t e r i o i d e s P - 6 0 .
Due t o
c i e n t tim e o n ly two c y s t i n e a s s a y s w e r e c a r r i e d o u t u s in g t h i s
H ow ever,, v e r y s a t i s f a c t o r y g r o w th c u r v e s w e r e o b ta in e d
la c k o f s u f f i ­
o r g a n is m .
( F ig u r e 6 , page 6 0 )
and t h e r e s u l t s a p p e a r t o b e i n good a g r e e m e n t w i t h v a l u e s o b ta in e d b y o t h e r
m e th o d s .
T a b le N o. V I , p a g e 6 2 , sh ow s t h e r e s u l t s o b t a in e d by t h i s m e th o d .
In a l l m ic r o b io lo g ic a l a s s a y s a v era g e r e s u lt s
from s e v e r a l l e v e l s
o f con­
c e n t r a t i o n m ust be u se d b e c a u s e o f l a c k o f u n if o r m it y o f th e g ro w th i n t h e
in d iv id u a l tu b e s .
C y s t in e D e t e r m in a t io n s
in
le c e r a l;
T a b le N o. IX , p a g e 7 4 , sh ow s th e c o m p o s ite r e s u l t s o f a l l c y s t i n e a s s a y s .
L -a r a b ln o s u s v a l u e s a r e show n i n red t y p e and i n d i c a t e t h e h ig h r e s u l t s ob ­
t a i n e d by t h i s m e th o d .
A l l r e s u l t s a r e a r r a n g e d i n c h r o n o l o g i c a l o r d e r sh ow ­
in g t h e r e l a t i v e a g e o f t h e h y d r o ly z a t e s .
T able I I I , p age 5 0 , g i v e s th e
tim e o f h y d r o l y s i s o f e a c h h y d r o ly s a t e and th e l e t t e r d e s i g n a t i o n u sed f o r
each.
A ssays c a r r ie d
o u t b y L u g g 's c o l o r i m e t r i c method and u s in g I e u c o n o s -
t o c g t e s e n t e r i o i d e e a r e i n f a i r l y good a g r e e m e n t .
On h y d r o ly s a t e N o. 25 and
N o . 2 4 , th e same v a l u e t e a o b t a in e d b y b o t h m e th o d s .
A - s m i t h i i a p p a r e n t ly
h a s p r a c t i c a l l y th e same c y s t i n e c o n t e n t a t b o th c u t t i n g s .
A -crisfca tu m sh ow s
e ^ B e v iT O jp X q
enTBA
but ^ eXa
©qx
eq»
e 8SB j9 ean^Bsi Baour eq>
jo
ut
-P P -
© 3b
eqq, q ^ M
so t ^ eXo
@ T% 4TT
vU e*.
b b t jb a
n j e sB e a o e p B qja T Je P
b
P er c e n t t r a n s m i s s i o n
S p e c t r a l T r a n s m i s s i o n Curve f o r P e t h i o n i n e
u s i n g the Csonka and Denton c o l o r i m e t r i c method.
S p e c t r o p h o t o m e t e r r e a d i n g s t a k e n a t SlCmp
-------- 1------
CM
10
8
S t a n d a r d H e t h i o n i n e Growth Curve
O
i
CD
I
Df Os l N NaOH
F ig
ure 7
I EUre
f o r L e u c o n o s t o c - m e s e n t e r o id e s F - 6 0 .
-
2
_
c
y
/
Z
t
4
H
+
"t
M
C o n c e n t r a t i o n o f m e th io n in e in
t"
4^
,
CT>
69
P er Cent M ethionine
2.40
Curve Showing the D ec r ea s e i n M et h i o n in e
V alu es w i t h t h e age of t h e h y d r o l y s a t e
A gropyron-iStnithii c u t 7 - 1 5 - 4 6 .
Time i n Days
F ig u r e 8
TABLE BO. VII
R e s u lts o f M eth io n in e D e te m in a t io n U sing Caonka and D enton’ s .Vethod
P r o te in e x t r a c t
D ate g r a s s was c u t
Iiy d ro ly s a te number
Date o f a s s a y
A - s m ith ii
5 »10-46
21
3—24—47
A-or is ta tu m
5—10—46 7 -1 5 —46
23
24
3 -2 4 -4 7 3 -2 4 -4 7
E -g ra o illis
8 -2 3 —46
8 -2 3 -4 6
8-A
8-B
1 -4 -4 7
1 -9 -4 7
V ita m in T e s t
C asein
6A
26
1 -3 -4 7
3 -1 8 -4 7
H a m e rste in
C asein
26
3 -1 8 -4 7
1 .6 0
1 .8 9
1.7 4
2 .3 8
2 .3 4
3 .0 7
3 .4 4
3 .3 4
1 .7 1
2 .2 6
2 .1 4
2 .2 4
2 .1 4
2 .6 3
3 .1 0
2 .9 5
1.86
2 .4 4
2 .0 5
2 .3 4
2 .0 6
3.12
2 .7 6
3 .0 0
2 .4 9
2 .4 0
2 .1 4
3.12
2 .4 7
2 .6 1
2 .1 0
3 .1 2
3 .0 9
3 .0 9
2 .4 6
Average V alue %
1.9 9
2 .3 3
2 .0 3
2 .3 2
2 .1 6
5 .0 1
TABLE HO. V III
R e s u lt s o f lr e t h io n in e a s s a y U sin g L e u c o n o s t o o - m e s e n t e r io id e s F - 6 0 .
P r o te in e x t r a c t
D a te g r a s s was c u t
H y d r o ly s a te number
D ate o f a s s a y
A v era g e V a lu e %
P r o te in e x t r a c t
D ate g r a s s was e u t
H y d r o ly s a te number
D a te o f a s s a y
A verage v a lu e %
A -e m ith ll
I-A
5 -1 8 -4 7
I-A
4 -1 0 -4 7
6 —10—4 6
I-A
4 -1 8 -4 7
21
5 -1 8 -4 7
21
2A
4 -5 -4 7 2- l a - 4 7
2 .1 8
2 ,1 8
1 .7 1
1 .7 1
1 .6 6
1 .7 9
1 .7 2
1 .8 8
1 .7 9
1 .7 6
1 .7 9
1 .8 5
1 .2 1
1 .2 1
1 .2 6
1 .6 0
1 .6 5
1 .6 6
1 .6 9
1 .6 9
1 .7 0
2 .5 7
2 .3 6
2 .5 7
2 .2 7
2 .2 6
2 .2 7
2 .1 2
2 .1 2
2 .0 2
2 .0 3
1 .9 9
2 .0 7
2 .0 9
2 .1 4
2 .3 2
2 .5 1
2 .3 4
1 .9 6
1 .7 8
1 .4 9
2 .3 1
2 .0 6
2 #33
7 -1 6 -4 6
22
Ba
4 -1 0 -4 7
4 -6 -4 7
2 .0 0
1 .9 0
1 .7 2
1 .8 5
1 .6 7
1 .9 0
2 .0 0
1 .7 2
1 .8 1
2 .1 0
1 .8 1
1 .8 6
A -c r i s t a t u e
5A
4 -5 -4 7
2 .2 2
2 .3 5
2 .1 0
2 .0 5
2 .1 0
2 .0 8
1 .9 8
2 .0 0
1 .9 8
2 .0 9
5A
4 -1 8 -4 7
1 .2 9
1 .3 8
1 .3 8
1 .7 6
1 .7 2
1 .6 0
1 .7 2
1 .7 9
1 .7 6
1 .6 0
6 —10—4 6
25
5 -1 8 -4 7
25
4 —5 —47
7 -1 5 -4 6
24
24
23
4 -1 8 -4 7 3 -1 8 -4 7 4 -5 -4 7
22
4 -1 8 -4 7
1 .9 9
1 .9 3
2 .0 6
1 .9 6
1 .9 0
1 .9 0
1 .6 2
1 .6 2
1 .6 2
1 .8 3
1 .8 1
1 .9 5
1 .8 5
2 .0 1
2 .0 2
1 .9 6
1 .8 0
H am erstein
C ase i n
24
26
4 -1 8 -4 7 4 -6 -4 7
2 .1 0
2 .0 4
1 .9 1
1 .9 1
1 .7 4
2 .5 9
2 .3 9
2 .3 9
2 .2 9
2 .3 5
2 .3 3
1 .3 6
1 .2 6
1 .3 1
1 .7 9
1 .7 6
1 .7 9
2 .7 0
2 .3 2
1 .6 8
1 .9 9
1 .3 6
1 .8 6
2 .0 8
2 .2 5
2 .1 1
2 .0 9
2 .1 1
1 .2 8
1 .3 5
1 .2 8
1 .4 3
1 .2 6
1 .4 4
1 .9 6
2 .5 6
1 .6 4
2 .0 7
2 .1 3
1 .3 4
3 .0 4
3 .0 4
2 »96
2 .8 1
2 .9 6
2 .8 4
2 .9 2
2 .9 5
5 .3 6
2 .9 8
72te e th io n in a D e te rm in a tio n U sin g Csonka and D e n to n 's M ethod.
No s p e c i a l d i f f i c u l t i e s Re re e n c o u n te re d w ith t h i s method and th e r e ­
s u l t s a p p e a r t o be q u i t e r e l i a b l e .
page 6 7 .
The s ta n d a r d cu rv e i s g iv e n in F ig u re 6 ,
The c u rv e i s a s t r a i g h t l i n e and i s r e p r o d u c i b le .
t h i s m ethod a r e g iv e n on T ab le V I I , page 7 0 .
The r e s u l t s of
A - c r i s t a t u a h as th e h ig h e r
m e th io n in e c o n t e n t o f th e g r a s s e s exam ined and i t was found t h a t th e m e th io ­
n in e v a lu e s d e c r e a s e w ith th e m a tu r ity o f th e g r a s s .
The v a lu e s o b ta in e d f o r
th e m e th io n in e c o n te n t o f c a s e i n a r e i n good ag ree m en t w ith each o th e r and
w ith v a lu e s g iv e n in th e l i t e r a t u r e .
B lack and B o llin g (9 ) g iv e 3.10% m eth­
io n in e in c a s e i n w hich a g re e s v e ry w e l l w ith th e r e s u l t s o b ta in e d i n t h i s e x ­
p e r im e n t.
M eth io n in e D e te rm in a tio n U sing L aucopostoc m e s e n te r io id e s P - 6 0 ,
No s p e c i a l d i f f i c u l t i e s w ere e n c o u n te re d i n grow ing t h i s o rg an ism on
s y n t h e t i c medium and few m o d if ic a tio n s w ere n e c e s s a r y in t h e medium p r e s e n te d
by B a rto n —! r i g h t ( 8 ) .
The a c id p r o d u c tio n of t h i s o rg an ism was n o t a s g r e a t
a s t h a t shown by L -a ra b in o s u s IT -S eQ h o w ev er, th e s ta n d a r d g row th c u rv e s ob­
ta in e d , F ig u re 7 , page 6 8 , gave v a ry sm ooth c u rv e s w ith l i t t l e v a r ia n c e b etw een
a s s a y tu b e s o f th e name c o n c e n tr a ti o n .
T ab le V I I I , page 7 1 , shows th e r e s u l t s o b ta in e d by t h i s m eth o d .
The
p e r c e n t m e th io n in e i n c a s e i n was s l i g h t l y lo w er th a n th o s e o b ta in e d by th e
c o l o r i m e t r i c m ethod.
A lth o u g h some v a r ia n c e was found b etw ee n d i f f e r e n t
h y c r o ly e a te s o f th e same p r o t e i n m a t e r i a l , much g r e a t e r d i f f e r e n c e s w ere ob­
s e rv e d i n d i f f e r e n t as.-ay s o f th e san e h y d r o ly s a te c a r r i e d o u t on d i f f e r e n t
d a te s.
The m e th io n in e v a lu e s d ro p o f f v e ry s h a r p ly w ith th e age o f th e
h y d ro ly s a te .
F ig u re No. 8 , page 6 9 , s h a r p ly i l l u s t r a t e s t h i s f a c t .
On T able
«*73 ■
I X , pa g e 7 4 , th e a s s a y s a r e a r r a n g e d i n o h r o n o lo g ie & l o r d e r and i t may b e
o b serv ed t h a t tiie r e
i s a g r e a t d e a l o f c h a n g e o c c u r r in g i n t h e m e th io n in e
c o n te n t w ith th e age o f th e h y d r o ly s a t e .
fa c to r st
T h is c o u ld b e due t o e i t h e r o f tw o
( I ) b a c t e r i a l a c t i o n d e s t r o y i n g th e m e t h io n in e j
may b e u n s t a b l e i n
( 2 ) th e m e th io n in e
th e h y d r o ly s a t e s o l u t i o n and be d e c o m p o sin g c h e m i c a l l y .
T h is ch a n g e i n m e th io n in e c o n t e n t ha$ n e v e r b e e n r e p o r te d i n th e
lite r a tu r e ,
p e r h a p s due t o l a c k o f o b s e r v a t io n on t h e p a r t o f th e d i f f e r e n t i n v e s t i g a ­
to r s con cern ed .
The h y d r o l y s a t e s w ere n e u t r a l i s e d t o a pH o f 6 . 3 and a u t o c l a v e d !m e d i­
a t e l y t h e r e a f t e r a t 16 pounds p r e s s u r e , I l S 0 C ., f o r 2 0 m in u te s th e n c o o le d
and c o v e r e d w i t h a l a y e r o f s u l f u r f r e e
t o lu e n e and s t o r e d
in a r e fr ig e r a to r ,
l a t e r m i c r o b i o l o g i c a l e x a m in a t io n u s in g s t a i n i n g t e c h n i q u e s r e v e a le d no
b a c t e r i a l g r o w th w h ic h w o u ld i n d i c a t e t h a t f a c t o r
p o n s ib le
f o r t h e l o s s o f m e t h io n i n e .
be h i g h l y d e s i r a b l e
( I ) was p r o b a b ly n o t r e s ­
I n v ie w o f t h e s e o b s e r v a t i o n s , i t w ou ld
t o make a more th o r o u g h i n v e s t i g a t i o n o f t h e c h a n g e s
w h ic h t a k e p la c e i n p r o t e i n h y d r o l y s a t e s .
P h a r m a c e u tic a l c o n c e r n s a r e new
m a r k e tin g p r o t e i n h y d r o ly s a t e f o r t h e r a p e u t i c u s e .
t i o n m ig h t h a v e p r a c t i c a l a s w e l l a s s c i e n t i f i c
T h u s , s u c h an i n v e s t i g a ­
v a lu e .
\
TABLE NO. IX
C o lo r im e t r ic
C o lo r im e tr ic
C o lo r im e t r ic
I
I - 3 -4 7
1 -4 -4 7
1 -9 -4 7
5 —16—4 7
3 -1 8 -4 7
3 -2 4 -4 7
4 —6 —47
4 -1 0 -4 7
4 -1 8 -4 7
-M
C o lo r im e t r ic
C o lo r im e tr ic
L -a r a b in o s u s
L ^ a r a b in o su s
L -a r a b In o eu s
C o lo r im e tr ic
C o lo r i i e t r i c
L -a r a b in o s u s
L -a r a b ln o s u s
L -m e s e n te r l o i d e s
Iz-ciesan te r i o i d e s
•M
1 2 -2 9 -4 8
1 2 -3 0 -4 6
2 -2 6 -4 7
3 —6 — 4 7
3 - 8 - 47
3 -2 0 -4 7
5 -2 1 -4 7
3 -2 5 -4 7
4 -1 0 -4 7
4 -1 4 -4 7
4 -1 9 -4 7
S
P r o te in
B a te R raes w as c u t
Number o f !E y d ro ly ea te
D ate
Method
D
Suffiti&ry T a b le S h ew in g A verage V a lu e s
A g r o p y r o n - s m it h ii
6 -1 0 -4 6
7-1 6 -4 6
28
22
I-A
2 1 2A
and C y s t in e i n
Jrass
r o te in
y d r o ly s a te s
A grop yron o r ist& tu m
B- - r a e i l l i s
6 —10—46
7 —16—4 6
8 -2 9 -4 6
23
48
24
SA
4A
6B
8A 88
A van Lge Vss lu e s f o r (I y s t i x -JS
Har e r s t e i n Case in
18A 188 26
0 .5 »
0 .6 2
1 .8 6
1 ,1 7
1 .8 8
1 .1 2
1 ,2 9
0 . 3 9 0 .3 9
1 .1 4
1 .0 1
1 .7 4
1 .5 2
1 .1 8
1 .1 1
1 .0 3
0 .7 9
0 .4 7 0 . 6 0 0 . 6 0
1 .3 0
i.* i§
1 .1 2 1 .3 6
1 .0 3
0 .7 8
0 .6 4
A vera ge Vsi l u e s f o r I a th i< )n in e
C o lo r im e tr ic
L -m e s e n te rio id e s
C o lo r im e tr ic
3>me se n te r lo i d e s
L - m e s e n te r io id e s
Iz -m e se n te rio id e s
2 .3 2
2 .1 6
3 .0 9
1 .9 5 2 .3 1 2 .3 3
1 .9 9
2 .0 6
1 .7 8
1 .8 6
1 .4 9
I . 56
2 .3 3
1 .9 6 2 .0 » 2 .3 6
2 .0 7
2 .0 3
2 .1 3
1 .8 0 1 .6 0 1 .6 4
1 .3 4
2 .9 8
76 T a b le IX , page 7 4 , sh ow s t h a t o f t h e g r a s s e s a n a l y s e d , t h e am ount o f
m e th io n in e and c y s t i n e v a r i e s w it h t h e v a r i e t y a s v e i l a s t h e a g e o f g r a s s .
A .-crifctatun ; h a s a h ig h e r m e th io n in e c o n t e n t and from t h e d a t a p r e s e n t e d ,
A - s m i t h i i a p p a r e n t ly h as a h ig h e r am ount o f c y s t i n e th a n th e o t h e r g r a s s e s
a n a ly s e d .
The more m a tu r e t h e g r a s s , t h e
a lth o u g h tn e r e
is
lo w e r th e c y s t i n e c o n t e n t and
l e s s d i f f e r e n c e , t h e m e th io n in e c o n t e n t a l s o d e c r e a s e s
■with th e a g e o f t h e g r a s s .
Tite m e th io n in e c o n t e n t v a r i e s b e tw e e n a b o u t 1 .9 6
&
and 2 .5 3 ; ; ; c y s t i n e
1 .1 4 t o 0 .7 9 % .
n u t r i t i o n i t b ecom es q u i t e
For a b e t t e r u n d e r s ta n d in g o f a n im a l
o b v io u s t h a t a m ore c o m p le te s t u d y o f ra n g e g r a s s
p r o t e i n s w ould b e h i g h l y d e s i r a b l e .
J . »<• E . In g g o f A u s t r a l i a and i . C»
C h ib n a ll o f E ngland h ave made v e r y v a l u a b l e c o n t r i b u t i o n s i n t h e s t u d y o f
t h e am ino a c i d c o m p o s i t i o n o f l e a f p r o t e i n s .
J . W. H. Lugg h a s made q u i t e
a th o r o u g h i n v e s t i g a t i o n o f t h e s u l f u r c o n t a i n i n g am ino a c i d s a s t h e y o c c u r
i n t h e g r a s s p r o t e i n s fo u n d i n A u s t r a l i a and some a t t e m p t h a s b e e n made t o
c o r r e la te h is
f i n d i n g s w i t h a n im a l n u t r i t i o n .
w ork h as b e e n d on e i n t h i s c o u n t r y .
As y e t v e r y l i t t l e
o f th is
Some r e c e n t d e v e lo p m e n ts M v e b e e n
made i n th e am ino a c i d c o m p o s it io n o f c e r t a i n d i e t a r y
p r o t e i n s and i t
is
t o be h oped t h a t t h i s w ork may b e c a r r i e d fo r w a rd and t h u s i n c r e a s e our
k n o w led g e o f p r o t e i n n u t r i t i o n .
—
V II.
1.
76 “
SOKMST
Sodium d o d e o y l s u l f a t e i s e s p e c i a l l y s u i t a b l e f o r th e e x t r a c t i o n o f
le a f p ro te in s .
High y i e l d s may be o b ta in e d by t h i s method and th e
f i n a l p r e p a r a t io n s c o n t a in a s h ig h a s 30% p r o t e i n .
2.
U n t i l a more co m p lete s tu d y h as b e e n made o f th e c y s t i n e m etab o lism
o f L a c t o b a c i l l l s a r a b in o s u s 1 7 -5 , t h i s m ic ro o rg a n ism sh o u ld be con­
demned f o r th e a s s a y o f c y s t i n e .
3.
Tlie a d a p tio n o f In g g *s m o d i f ic a t io n o f th e F o lin - K a r e n s i m ethod f o r use
w ith th e Coleman s p e c tr o p h o to m e te r g iv e s c o n s i s t e n t r e s u l t s w hich a r e
in good ag ree m en t w ith v a lu e s o b ta in e d by o th e r m e th o d s.
4.
recovery o f c y s t i n e added t o th e p r o t e i n h y d r o ly s a te was found t o be
95-98% u s in g Luggt S c o l o r i m e t r ic m e th o d .
I t was a l s o found t h a t a s h ig h
a s 13% o f th e c y s t i n e was d e s tr o y e d by a c id h y d r o l y s i s .
b.
le u c o n o s to c - K e s e n te r io ld e s p-6 0 i s v e ry s a t i s f a c t o r y f o r th e m ic ro b io ­
lo g ic a l a ssa y of c y s tin e .
6.
The e s t i m a t i o n o f m e th io n in e by Csonka and D en to n ’ s m o d if ic a tio n o f th e
McCarthy S u lliv a n method i s v e r y s a t i s f a c t o r y and th e r e s u l t s a r e in
good ag reem en t w ith l i t e r a t u r e v a lu e s o b ta in e d by o th e r m e th o d s.
7.
Ia u c o n o s to c - ir e a e n te r io id e s P-60 may be w e ll ad a p te d f o r t h e m ic r o b io lo g i­
c a l a s s a y o f m e th io n in e .
8.
'e t h i o n i n e i s u n s ta b le i n p r o t e i n h y d r o ly s a te s p re p a re d and s to r e d u n d e r
s t e r i l e c o n d itio n s .
The lo s s o f m e th io n in e in such h y d r o ly s a te s i s p r o ­
p o r t i o n a l t o th e ag e o f th e h y d r o ly s a t e .
9.
The m e th io n in e and c y s t i n e c o n te n t o f M ontana range g r a s s e s v a r i e s co n ­
s i d e r a b l y w ith th e d i f f e r e n t v a r i e t i e s a s s a y e d .
77
(Summary C o n tin u e d )
10.
The am ount o f m e th io n in e and c y s t i n e i n th e g r a s s e s a n a ly s e d w as fou n d
t o be c o n s i s t e n t l y
lo w e r i n t h e more m ature g r a s s .
The a a r l y s p r i n g
c u t t i n g s show a h i g h e r p e r c e n t a g e o f b o th m e th io n in e and c y s t i n e .
-78 -
viii.
ACKno isnoassaiTS
The a u th o r w is h e s t o e x p r e s s h is a p p r e c i a t i o n t o D r. Leon H . Jo h n so n
who a id e d i n o u t l i n i n g th e p roblem and gave many c o u r ts u* swmU h e l p f u l
s u g g e s tio n s d u r in g th e p r o g r e s s o f th e i n v e s t i g a t i o n and i n th e p r e p a r a t io n
o f t h i s t h e s i s ; and a l s o to P r o f e s s o r P . C . S a in e s and D r. K a rl F . S w in g le ,
f o r th e tim e a s s i s t a n c e and a d v ic e g iv e n him d u r in g th e y e a r w h ile t h i s work
was b e in g c o m p le te d .
IX .
LITERATURE CITED
(I)
A lb a c e s e , A. A. , I n d . E ng. Chem. Anal . Ed. 1 6 , 609
(2)
B a e r n s te i n , H. D ., J . E lo l . Chem. 3 9 , 125 (1 9 3 0 ).
(3)
N
W
(4 )
W
t»
w
w
n
M
It
It
H
9
H
W n
n
w
(6 )
(6 )
"
f ! ' 663 (1 9 3 2 ).
"
M l . 4 5 1 , (1 9 3 4 ).
"
3 3 , (1 9 3 6 ).
2 5 , (1 9 3 6 ).
(7 )
B a i le y , K ., Blochem . J .
1396, (1 9 3 7 ).
(8 )
E a rto n -W r ig h t, The A n a ly st 7 1 , 2 6 7 , (1 9 4 6 ).
(9 )
B la c k and B o ll in g , The Amino Aoid C o m p o sitio n o f P r o te in e and F oods,
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