Percrystallization of cystine and tyrosine in the presence of inorganic salts
by William D Saxton
A THESIS Submitted to the Graduate Faculty in partial fulfillment of the requirements for the degree
of Master of Science in Chemistry
Montana State University
© Copyright by William D Saxton (1955)
Abstract:
A study was made of the percrystallization of cystine and tyrosine from a wool hydrolysate across a
cellophane membrane in the presence of inorganic salts. The percentage -of these two acids passing
through the membrane was calculated. BBRCBYSTA&LIZATION OF OYSTIBB ABD TXROSIBB
IE TEm PRB3EB0E OP IBORGABIO SALTS
'
WILLIAM D, 8AXT0R
A TRESIS
,
S u b m itted to th e G rad u ate F a c u lty
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 eg ree o f
M aster o f S cien ce i n C hem istry
at
Montana S ta te C o lleg e
Approved $■
Head5 M ajor Department.
ChairmMis Exam ining Oommittee
Bozemans Montana
Ju n es 19^5
S a. I
" 2”
2-
TABLE OF CONT • NTS
ABSTRACT .................................................................................................................................................3
INTRODUCTION ...............................................................................................................................
EXPERIMENTAL PART
^
1 .................................................................................................................. 7
. . . . .
7
R e s u l t s ................................................................................ .... . . .
26
Methods . . . . . . . . . . . . .
..................
EXPERIMENTAL PART I I ............................................................................................................ 3 ^
D I S C U S S I O N ......................................................................................................................................... 38
SUMMARY.................................................................................................................................................... \±6
LITERATURE C I T E D ............................
!48
ACKNOWLEDGEMENT............................................................................................................................. J4.9
ABSm&gf.
A s tu d y Wds made of; th e p e p G r y s t a l l i z a t i o n o f o y g tin e and
ty r o s in e Arom a wool h y d r o ly s a te a c r o s s a ' c e l l ophsme 'membrane in
,th e W e'senee o f ln o r g a n i e , .S a lts 0 th e p e rc e n ta g e of th e s e two '
•a c id s 1.p assin g through : th e ' membrane,, was c a lc u la te d *
''
V
IBBROmOTIO#
Kobe? (?) n o te d t h a t i f one e n c lo se d a l i g n i d in- a c o l l o ;,dlon bag and suspended such &■bag over a f I ame "o r" e l e c t r i b
h e a te r , ev a p oration took p la c e eo r a p id ly th a t I t #as' v i r t u a l l y
IapoaA ible t o ra la d the liq u id tie th e b o ilin g p o in t,
#vapo*
r a tio n ta k e s p la c e on a i l s u r fa c e s , as though"a b a ll o f w ater
was suspended in a ir w ithout a co n ta in in g membrane;
sig n a ted t h is phenomenon p erv a p o ra tio n .
Kober de­
D i s t i l l a t i o n by t h i s
means i s c a l l e d p e r s t i l l a t i o n and may be c a r r ie d " o u t by s u s ­
pending the c o llo d io n bag i n a c o ld b o t t l e and h e a tin g th e ' 1•
l i q u i d i n s i d e th e bag' e l e c t r i c a l l y o r w ith steam .
I f th e liq u id '
c o n ta in s & so lu te * the s o lu te w i l l d if f u s e through th e membrane
w ith th e l i q u i d and rem ain oh the s u r f a c e o f th e b ag "lh " c r y s t a l
form , ' ih e te rm f o r t h i s phenomenon is - p e r c r y s t a l l i s a t i o n ,
Kqber found th a t c o l lo id s do not d if f u s e through th e madbrank
and Suggested th a t c r y s t a llo id s cou ld be separated'from e o l l o i d a l c o n ta m in a te s by p e r c r y s t a l l i z a t i o n ,
he succeeded i n
c r y s t a l l i s i n g h is t id in e , on th e su rfa ce o f a c o llo d io n bag con­
ta in in g a p r o te in d ig e s t io n r e sid u e in stron g h y d ro ch lo ric acid*
Oeilophane c a sin g s were used in p la ce o f c o lIqdlon by
K arber (3) to con cen tra te v e r y .d ilu t e : p r o t e in s o lu t io n s w ith
th e sim u lta n e o u s rem oval of s a l t s by pep-vapor a t io n akd'-pdiS
c ry s ta llis a tio n ,
He hastened parvaporatlon by u se o f an e l e c -
t r l c f a n r a th e r than h e a tin g th e bag.
Gortner (4) su g g ests
t h a t c e llo p h a n e i s p r e fe r a b le t o c o llo d io n membrane f o r p e r -
v ap o r& tln g .
Holmes (6) b r i e f l y o u t l i n e s p e r v a p o r a tio n methods
. v■
and t h e i r u s e s . • T a iilo' GhimaraSs da F en aeca ( 5 ) d iscu ssed - p e r*
vapor a t io n a p p a ra tu s ' and o p e ra tio n s *
^
-
. S hat th e m ethod h a s been g e n e r a l l y ' o v e r l bdked Is. e l e a r l y .
l l l u s t r a t b d by th e f a c t that" th e -a b o v e mentioned w orkers -,aneth e o n ly ones l i s t e d 'In ' th e l i t e r a t u r e a'S -having nsed- -.peh^-'
•
.
'
■
■
''"I ' v a p o r a tio n m eth o d s,-'
-
•"
!Phe p o s s i b i l i t y o f q a a l i t i a t l v e and q u a n tita tiv e aepar^
a t io n o f amino a c id s from-a- p r o t e i n d ig e s tio n ' r e sid u e by p e r*
c r y s t a llis a t io n ' on. a cello p h a n e c a sin g was explored* ' A.: por*
t lo h o f th e p r o te in d ig e s t ob tain ed from the h yd roch loric' a c id
h y d r o ly s is o f ca sein # and su b seq u en tly p a r t i a l l y n e u tr a liz e d
w ith sodium h y d ro x id e , was p la ced i n a c e llo p h a n e basing*?'. A f te r
th ree days an e n c r u s ta tio n was observed on th e su rfa ce of, th e
easing^
M o r esc ep id a n a ly s is r e v e a le d th e p resen ce o f sodium
c h lo r id e i n la r g e q u a n t it ie s ,
Sm all amounts o f c y s tin e and
ty r o s in e - i n c r y s t a l l i n e fo rm ■were o b se rv e d a l s o .
'
I
A p r o te in d ig e s t f r e e o f in o rg a n ic s a l t s W s prepared by
adding barium hydroxide to s u lf u r ic a cid h yd rolysed ca sein *
w ith subsequent f i l t r a t i o n 1to- remove th e b ariu m su lfa te *
When
th e s o lu t io n was f r e e b f barium and s u lf a t e ions# i t was p la ce d
in c e llo p h a n e easln gS v
Ko c r y s t a l l i s a t i o n took p la c e bn th e
- su rfa c e # ev en When h e a t was a p p lie d ,
Q o llo d lo n bags in th e shape o f te n in ch t e s t tu b es were
f i l l e d w ith th e same p r o te in d ig e s t so lu tio n *
A g a in / ad' c r y s t a l
a.
*6*
l i z s t t i o t t r e s u l t e d * ' ev en w ith h e a t ap p lied # ■
Thus I t 'was e v id e n t t h a t amino a s id e and p e rh a p s a l l ' othdtt
. o rg a n ic eomp'outtds w i l l ' n o t 'p a ss th ro u g h c e llo p h a n e o r c o lI o d i on
membranes by p e r o r y s t a l l i z a t i o n m ethods i n th e -absence o i 'i n n
o rg a n ic s a l t s ^
T h erefo re* i t was d e c id e d to l i m i t t h e .r e s e a r c h
t o th e d i s t r i b u t i o n o f c y s tin e and ty r o s in e a c r o s s -a -c e llo p h a n e
membrane from .a p r o t e i n h y d ro ly sa te , c o n ta in in g in o rg a n ic ’s a l t s *
The e x p e rim e n ta l work i s ' d iv id e d i n t o two p a rts *
P a r t . I c o v e rs
th e m easurem ent o f th e amount o f - c y s tin e and ty r o s in e p a s s in g
th ro u g h C ellophane membranes from- p r o t e i n h y d r o ly s a te s con*,
t a l n i n g ammonium s u lf a te * ammonium c h lo r id e , and sodium c h lo r id e
re s p e c tiv e ly .
P a r t I I d e s c r ib e s th e measurement o f c y s tin e and
ty r o s in e p a s s in g fro m p ure so lu tio n s- o f th e s e a c id s In th e p r e ­
sence o f t h e above l i s t e d .s a l t s , a tte m p ts .to p e r c r y s t a l l i s e
s o lu tio n s o f amino a c id s a lts * and e x p e rim en ts w ith , c o llo d io n
membraneSe
-
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r?*
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- - i . ..
. . - pmpAB4?%oB o#:?ao^iB.m ®Ra,% aATss.:..
- ;: -.
.. ' /. ....
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r.
. A tw e lv e l l t e i ;
e& teaetea wool
..
m w es,
.../
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,
W
.s ix .’l itep@ ':of..6H
Titlofi wae, d ig e ste d f o r
.
wl%h, . , alqeb. 9l^
w e e added„
h o q ^ s, o ver ,a low flam es
s o l*
% e b lM k
e o l# lo m . pbtalRe^ # 8 deool w ip e d by W lll o g I t for- tNro Wmfe
w ith E o f lt d eo o lo flp lA g :.oapboo*
Pooled, end' e u o tlo ^ . ^ l l w . \
t e r e & . $he treatm ent was fe^eated" .th fe e .times*. .p ro # a l# & * ,.
n ea rly e o lo r le e g solTitlon*. A, o e g a tiy e , B iu ret t e a t proved,.t&at
the p r o te in had .been, co m p letely -broken down .to amino- aolds.0...
ih e s o lu t io n was .n e u tr a lis e d t o .Oongo-Bed by %
th e a d d itio n o f
6# SHjjOBa
A sm all q u a n tity of.
,was th e # added t o ,pro*,
v en t th e o r y s t a l l l s a t i o n of: ey a tln # and t y r o s i n e ,. # i o h oooura
h t a pH o f 3 t o
.th e end point) ,o f #ongo,.Red*
._
- -
. ..
A h ydroohlorlb .a p id -d lg e e tM ..Oaseln'rea id n e .w48;#rep#P&d
by t h e method ,d escrib ed above* u s in g
HG!. In p ia e e o f
When a ol.ear s o lu t io n was o b tain ed end had , g iv e n a n e g a tiv e .
■
.
I
■*
J , . .1 .
.
I
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' .
H
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'l• „
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<
B iu r et te st* . I t was d iv id e d In to two. p o r tle ts * one o f vbioh was
c o n sid e r a b ly la r g e r than th e other^
The la r g e r p o r tio n ,v a t
n e u tr a lis e d t o QongoJSed by t h e a d d itio n o f
. sm a ller p o r tio n was n e u tr a lIped .b y ,
EHuBH» . The:
,
.
EaGH^ - A gain t o p revent
th e c r y s t a l l i s a t i o n o f c y s tin e an# ,tyrosin e* a sm all q u a n tity
o f HGi was add©eh;
■
•.,
■ • ■
FmBORY&%a&&I2AT%OB ABp OOB&BGTIG# OF W C m # # # # ;
'/':.. ^wo ha&dred m i l l p o r t i o&e o f tb o (m ^)^SOky ^ h G l ,
■ ''
■
'
■
I -1V1 V-‘ ,'
and lSaGl c o n ta in in g h y d ro ly sates were placed, in cellophane
■
■■
'
'
'
e a sin g g w hich w ere l e f t h an g in g i n a i r a t room, tem peraim ne f o r
ijB hours*
$he i l l u s t r a t i o n s o n th e fo llo w in g p ag es .show th e
developm ent o f e n c r u s ta ti o n ah v a r io u s sta g es# . T h e c a s in g on
th e l e f t i n f i g u r e I was p h o to g rap h ed a t s i x hours# ■Ho per*,
o r ys t a l l I Emt i on, can be o b serv ed a t t h i s stage#.
The c a s in g a t
th e r i g h t i n f i g u r e I shows th e e n c r u s ta ti o n d ev elo p ed a t t h
hours#
Both o f th e s e c a s in g s c o n ta in th e (HH^)2SQ^ s o lu tio n #
F ig u re S shows th e : developm ent o f e n c r u s ta tio n a t 12 h o u rs on
a c a s in g c o n ta in in g 100 ml* o f HHLOl s o lu tio n #
Observe t h a t
a s w ate r p e r.v a p o ra te s and th e volume d e c re a s e s , pe r e r y s t a l *
l i g a t i o n p ro c e e d s from th e .' to p downward fo llo w in g th e l i q u i d
le v e lo
A p o in t Is , reac h ed when th e volume i s re d u c e d approx**
im a te ly by one h a l f and th e l i q u i d becomes v isc o u s*
At t h i s
p o in t p e r c r y s t a l l i z a t i o n b e g in s f o r some obscure, r e a s o n .a t th e
bottom #
From t h i s ' tim e on,, th e volume d e c re a s e s v e ry l i t t l e .
and p e r c r y s t a l l i s a t i o n p ro c e e d s u n ifo rm ly th ro u g h o u t t h e .s u r ­
fa c e a r e a o f th e liq u id #
I 1Wo
c a s in g s c o n ta in in g th e (HE^)g$0^ s o l u t i o n a r e shown
in f ig u r e 3 *
The c a s in g on th e r i g h t i s a t th e 2i| ho u r sta g e
and th e c a s in g a t th e l e f t i s a t th e Ij-B h o u r s ta g e o f develop*
ment*
i t may be o b serv ed th at t h e r e i s l i t t l e
d if f e r e n c e i n
th e developm ent, o f e n c r u s ta ti o n on th e two c a s in g s , a lth o u g h
th e lj.8 hom?, c r u s t I s .th ic k e r th ro u g h , th e ,middle,*
... J iIgurejLf. i s .a e!L.ose*up-.of th e W h o u r cruet*. .Obeerye
t h a t th e o u te r la y e r-.o f o r u s t i s Uraeleehjs.,, T h is -was ehrased. .
.p ro b a b ly .b y -th e , p r e s s u r e -e x e rte d .from t h e . in s id e where - f u r th e r .
p e r e.ry s t a l I i za t l on w a s -ta k ih g .place*
A fte r IfS h o u rs ue-
f u r t h e r p e r c r y s t a l l i s a t i o n , w i l l ta k e p la c e u n le s s th e c r u s t i s
removed^ in . w hich c a se a sm all - q u a n t i t y of' new c r u s f a t i o h :■
fo rm s*- The e n c r u s ta ti o n m easures .ab o u t seven am*., a t th e max*-.,
imam, p o in t .of th ic k n e s s * .. 1
.
.. .
figu re 3
/3 -
fij+ te 4
''.I?.-.".
'
'-'V p ie c e
e n c r u s t a t i o n was. c o l le c te d by p la c in g a l a r g e ;c le a n
of
p a p e r'U n d e r t h e . c a s in g and s c ra p in g th e c r y s t a l s o f f.
.-'! W ith & .Spatnlkii
!%e e h c r h s t a t i o n aro u n d ,.the i l q u i d eelm ^n
■' was e a s i l y d e ta c h e d fro m th e c a s in g as i t came o f f .d e a d ly in .
lar&a. chunks*
Above th e liq u id column th e c a s in g wan, d ried
and th e e n s r u s ta tie n Adhered to th e membrane and had t o be
Scraped o ff*
A f te r ' a l l o f the e n c ru s ta tio n had been scraped
o f f th e c a sin g and p la ced In a b ea k er, the casing: was punc­
tu red
and
th e l i q u i d d rain ed in to a beaker*
The e a sin g was
th en cu t open and th e e n c r u s ta tio n was scraped from th e in n er
surface*
la r g e c r y s t a ls o f in o rg a n ic s a l t s were .formed ok th e
in s id e r*
Ihe o u tsid e e n c ru s ta tio n was sawed fo r measurement of
c y s tin e and ty rb a in e con ten t by methods to be described*
fu r th e r uae was found f o r the liq u id and e n c r u sta tio n from
th e in sid e* as th e t o t a l co n ce n tra tio n o f c y s tin e and tyro*-
Sine in th e e a sin g was measured b efo re hand*,.
O olorlm et r i o t e s t s f o r e y s tiiie amfi -ty ro s in e w e v e s e l e c t e d
' e$i th e b a s i s ©.f" t h e i r a d a p t a b i l i t y to tiae" I h th e ' S ^eO tro p h o t . m eter*
fI o y o d a f S. ad ap tio n ^ o f 'th e Flem ing re a c tio n . ( I ) :.®as
. chosen f o r th e a n a l y s i s o f cystin e*'
One m i l l i l i t e r o f c y s tin e -
. c o n ta in in g s o l u t i o n o r one gram o f c y s tin e c o n ta in in g ■s o li d
.•
" is . p la c e d i n a g ra d u a te d ...c y lin d e r w ith £0 mg* o f . t i n g d u s t
■
. -'fv '
. and. S mi.# o f I S Hdlo S ew n and o n e - h a lf ml* of' dimothyl^p^phenylene diam ine h y d ro c h lo rid e re a g e n t fo llo w e d by #,5 . ml*, o f
f e r r i c alum a re added im m ediately*
t o 15 m
Whe s o l u t i o n is- d i l u t e d
l stoppered* and r e a d a f t e r 12 h o u rs a g a in s t .a
•' s ta n d a r d p re p a re d I n th e same way* H y s tin e i s re d u c e d b y s in e
i n HCl and th e r e s u l t i n g ' c y s t e i n e . is- warmed w ith cU m sthyi-p-.
p h e n y lene diam ine h y d ro c h lo rid e ' i n th e 'p re s e n c e o f f e r r i c io n s
. and -a b lu e c o lo r develops*..
F o r th e a n a ly s is o f ty r o s in e Zuwerkalowf s m o d if ic a tio n o f
t h e . M illon^W eiss r e a c t i o n ( 2 ) was chosen#
Whe method c o n s i s t s
■o f d is s o lv in g 10 mg * o f p r o t e i n o r p r o te in d ig e s t i n one mSU
o f £fo WaOHy a n d ad d in g .J ml# o f IO^ a c e t i c a c id , 2 ' ml* o f l W
HgSOt ( iu
and one drop o f a$ $ WaWOg» : I h e s o lu tio n
i s l e f t s ta n d in g f o r a n a r b i t r a r i l y s e le c te d l e n g t h o f tim e
■and r e a d a g a in s t a s ta n d a rd w hich was read , a f t e r th e Same '
le n g th o f tim e*
W nlike th e W illo h r e a c tio n * C h lo rid e io n s do
n o t i n t e r f e r e i n t h i s r e a c tio n #
; 'le a d s t o h ig h r e s u l t s , however*
Whe p re se n c e o f try p to p h a n
Whe t e s t i s s p e c i f i c ..f o r th e
>i6>-
■phenolic hydroxy group in th e presence of Hg*9. H g ^ 5. and W t
Ie n ea. producing a,,b rick red. c o lo r.
I f th e so lu tio n ' is- Heatedii.
th e , color, develops rap id ly* hut a "p r e c ip ita te ' soon develop0"1"
iw h ieh d ecreaset th e ■v a lu p . o f' th e t e s t •as a a u h a tl^ a tiv e ,,pro*/'
V'..
: \.
-
...
,
.
‘
. vhr.' •"'■
'
I f th e sd lu tih n i e l e f t a t room tetiperaturOa th e color
.develops slowly* ■hut ■does not p r e c ip ita te n e a rly i s ' rapidly^. ■■
.A ll tyrosinie. t e s ts . in t h i s re se a rc h vend' read a f t e r HO # !n u te s
a t.rd o m .teiR p eretu re* .
-'... ;
-
-
. - . - . ' /. '
. P r e p a r a tio n , o f R eu g en ts
M m © thylgp»phenvlene diam ine h ydro c h i o r I d s 0 ■f i v e hundred
m illig ra m s .o f d im e th y lrp -p h e h y lene diam ine h y d ro c h lo rid e were
. d is s o lv e d i n .a c o o le d -m ix tu re o f 100 mOU o f w a te r and 0
o f c o n c e n tr a te d
lite r*
F e r r i c Alum*
.Water whs added t o a -volume o f one
. ;
$wenty*-five grams o f F©2 (S0^ )^ o 2ifH20 were d l s ,
so lv e d i n 100 ml* o f HgO and H mU o f c o n c e n tr a te d HgSG^0
fh e
s o l u t i o n was d i l u t e d to 200 M 0
-M ercu ric S u l f a t e * .. T w en ty -six m i l l i l i t e r s o f c o n c e n tr a te d HgSOh
-were d i l u t e d t o 500 ,ml*.,and $0 gram s o f HgSOju were, added*
S p e c tro p h o to m e trib A n a ly s is
r'
'
'
'
The Beckman lfrBfr sp e c tro p h o to m e te r was th e in s tru m e n t
.
chosen f o r th e a n a l y s i s , Micham (8) in d ic a te d t h a t #8o#H90mu
i s t h e . w ave;l e n g t h , a t Which t h e c y s tin e t e s t i n th e Flem ing
r e a c t i o n h a s maximum a d s o rp tio n *
A s e a rc h o f th e l i t e r a t u r e
■*>17""i"
f a i l e d to r e v e a l any d a ta on th e wave le n g th f o r maxinmm
a d s o rp tio n f o r th e M illon-W eiss re a c tio n *
T h e re fo re , th e
p ro p e r wave le n g th had to he found by p l o t t i n g tr a n s m itta n c e
a g a in s t wave le n g th f o r .th e c o l o r 1developed by th e ty r o s in e
tests
■.
''
■
A gt& ndard was .p re p a re d c o n ta in in g #lg<j o f ty r o s in e p e r
ml#.
The r e a g e n ts were added and th e s o lu tio n was l e f t f o r ,,
«
:.
y
30 m inutes* A b la n k c o n ta in in g th e r e a g e n ts b u t no ty r o s in e
was a ls o p rep ared * ' ,The tr a n s m itta n c e a t wave le n g th from lf.00
to 750im w ith in c re m e n ts o f 2^mu w ere read*
F i r s t a Water
b la n k was p u t in to th e sp e c tro p h o to m e te r and th e tr a n s m itta n c e
d i a l a d ju s te d to z e ro , th e n th e ,,reagent b la n k was p u t 4 n and
a g a in th e tr a n s m itta n c e d ia l a d ju s te d to z e ro , th e n th e
,■
•
,
■
'« ■
s ta n d a rd .w a s p u t in and th e tr a n s m itta n c e was read* T his p ro ­
c e d u re was fo llo w e d ,at each su c cee d in g wave length®-
Wave
le n g th was p l o t t e d a g a in s t tr a n s m itta n c e in f ig u r e 5? re--v e a lin g -a minimum tr a n s m itta n c e a t
w hich i s th e wave
le n g th f o r maximum a d so rp tio n #
.
S ta n d a rd C o n c e n tra tio n Curves f o r C y stin e and T y ro sin e
A s e r i e s o f c y s tin e and ty r o s in e s o lu tio n s o f v a ry in g con­
c e n tr a tio n s were p re p a re d and t h e i r t e s t c o lo r s were developed#
Curves w e re . drawn p l o t t i n g tr a n s m itta n c e a g a in s t c o n c e n tr a tio n ■
u s in g a wide ran g e o f c o n c e n tra tio n s *
The ran g e of concen­
t r a t i o n in w hich th e cu rv e m ost c lo s e ly .a p p ro a c h e s a s t r a i g h t
l i n e was chosen a s th e c o n c e n tr a tio n ran g e f o r th e s ta n d a rd
' ' ' The most s e n s i t i v e .range f o r c y s tin e s a c fo u n d ;t o Ce
from .« 1 0 -to
m g ^ - p e r ’ ■A ' s ta n d a r d cu rv e w i t h ; cenc^jk*t:
tr a tlo iis WithAh•t h i s 'range> and w ith increments- of- vdiS ''mge■ p e r ml ^ was p re p a re d *
curve* ••
' - ' ■■■
BsI g u re 6 I s th e s ta n d a r d c y s tin e
'
'•
'
.
-- ' - ,
From' alS to * 3% mg* p e r mi* was fo u n d to be th e m o st'
e f f e c t i v e ra n g e f o r th e ty r o s in e t e s t -w ith in c re m e n ts o f
»02 mg* p e r
fig u re
• th e s ta n d a rd ty r o s in e cu rv e l a shown in
-=
'
'
H a! en o so S3 -a
D H» toaa!>i-it4H.«taa5>iaHi
19-
wave le n g th In mu
%AVE LENGTH AT MIRI MOM TRANSMITTANCE FOR M E TYROSINE TEST
fig u r e 5
►3 Bi R O SG to M
9 H*
to0 2 5 >t-3 »-3 HSeC/3 B ; > ® H 3
IOQ
Uc
3C
10
713
720
c o n c e n tr a tio n
CYSTINE STANDARD
I n m g /m l
CURVE
fig u r e
725
6
at
mu
730
100
Es W -aj 55 CO^ H El
«C S O W
I
ro
-H C
H
I
As Cfl « O M a E-t
Uo
30l
733
________________________________________________________________
720
72$
c o n c e n tr a t io n in mg/ml
TYROSINE STANDARD CURVE a t
fig u r e
7
Itf 5
TJo
nai
TJJ
-22m
Q u a n tita tiv e A n a iy a ie o f th e S h e g u s ta tio n s and F r o te ih
H y d ro iy sa te S o lu tio n s: f o r S y s tin e and T yrosine
I n o rd e r t o f in d sample s iz e s c o n ta in in g co n cen tra tio n s
o f c y s tin e and ty r o s in e w ith in th e c o n c e n tr a tio n range- of th e
c y s tin e and ty r o s in e standards* i t was n e c e s s a ry to make--nu­
merous t r i a l r u n s o
I t was found t h a t one gram of: - s o lid out#
s id e m a te r ia l '-and @1 #&<, o f I n s id e s o lu t io n c o n ta in e d concen­
t r a t i o n s o f c y s tin e w ith in the. .range on th e s ta n d a r d cu rv e %
%be game q u a n t it ie s were found t o be s u ita b le fo r , th e ty r o sin e
te s t *
j,.
■ "
,
Seven bags c o n ta in in g th e p r o t e i n h y d r o ly s a te ; w ith :
(MH^)g80L* seven co n ta in in g
w ere analysed*
and four c o n ta in in g
fhe- t o t a l c y s tin e and ty r o s in e p r e s e n t in
200 mle -of ea c h o f t h e above s o lu tio n s w ere c a lc u l a te d by
a n a ly sin g *1 #&* sam ples o f each so lu tio n *
Three t r i a l # were
,.run- i h each ,case and th e average- v a lu e -was taken* ' T able, I
show# th e c o n c e n tr a tio n o f c y s tin e and ty r o sin e in th e
8 0 k . so lu tio n * Tablq I I in the. BB^Gl ab lution * and Table I H 1#
th e ,. I a d l s o lu tio n * ;■
•
,-,
, The e n c r u s ta ti o n on each bag was c o lle c t e d and weighed*
One gram samples from each bag were analysed f o r c y s tin e and
ty ro sin e*
The t o t a l q u a n t it ie s o f c y s tin e and ty r o sin e in the
su rfa ce e n c r u s ta tio n were then ca lc u la te d *
The p ercen t o f eye*
tin e , and ty r o s in e p a ssin g through th e membrane was th en c e l*
eu la ted f
,
, . - 23 -
$ a b le IA
O o ao e & tratio a o f ^ y n o sin e i n a H ydnolysate'
C o n ta in in g (HH^)^SOr
R eading
Cone« p e r
sam ple ( . 1 m l,)
Gone,
p e n . ml
8 2 ,2
,2 2 2 mg,
2 ,2 2 mg.,;
200 m l*-
82
,226
e,%G .
200-':
Bi
, 2l|4
2 blj4
200
o.
U o tal
Volume
T o ta l
.U y m sin e
W k $8*
# 8
■«**=*.
a v e ra g e t o t a l co n e, o f ty r o s in e i n 200 soli, o f s o lu tio n . Ij61 mg.
T ab le IB
C o n c e n tra tio n o f G y stin e i n a H y d ro ly sa te
G o n tain in g ■(
. R eading .
Cone, p e r
. sam ple C.X mDU)
Gone,
p e r ml,.
T o ta l
Volume
T o ta l
C y stin e
7k
,2 7 0 mg.
2^7.0 mg.
200 a f ,
^%0 mg*
-^3 - . ' -
*29%.
2.8%
200
268
*2^8
2^8
200
‘
#6
VW
=ItiW
'
a v e ra g e t o t a l cone, o f c y s tin e i n 200 ml,, o f s o lu t io n
Sijf mg.
. Table IZA
.
I
C o n c e n tra tio n o f T y ro sin e in a H y d ro ly sa te
' '
C ontaining m rO l
y.'/':'
'
Gone, p e r
sample ( ,1 m is)
Oone*':
p e r mid, ,
T o ta l ,
Volume
&817
2 t l7 mg*
300 # 4,
&d!8
200i\'''^K-
241?
200/
,218
.
-
:
" ■
:*217-
\
., T o ta l ..
• "T v ro sine
-H A
‘ ■
.
- ' '
(■'.average t o t a l co n e, o f ty r o s in e in 200 ml* o f a o ln t i o n ,# ^
i
T&bla ZZ B
O o n e e n tra tio n o f 'Oya t i n e i n a H y d ro ly sa te
C o n ta in in g HHiQl
H eading
Gone," p e r
sam ple C, 1 ' ml o) .
Qo n e * ""
p e r m l.
T otal
Volume
T otal
Q ystine
7#
*M6 o mg^
2*6,0 mg*
200 mle-
#20 mg*
*260
2*60
200
#20
&2#8
2*##
200
' ' #16
. .7# - - - '
av e ra g e t o t a l conc» o f c y s tin e i n 200 bi1<, o f s o l u t i o n
#18,7 mg.
mable IIIA
O o n o e n tra tio n o f T g ro sin e i n a H y d ro ly sa te
C ontaining BaGl
Reading
Gone,- per ■
sample (»1 ml a")
81
8 1 *# .
mg;
,2W
81*2
Total
^yrosine
Gonofr-
T otal
2;lj.6 mgv
200
2*^0
200
Mo
2;i),0
200
Mo
Volnme
pep a i f
average t o t a l eonoi of ty ro s in e in 200
rtili of sO lntion
M b mg*
T able I I I B
C o n c e n tra tio n o f C y stin e i n a H y d ro ly sa te
1
C o n ta in in g BaGl
T o ta l
. Volume
T o ta l
C y stin e
R eading
Gone# p e r
sample U l mU ).
Genoa
p e r nA ;.
73
p286 mg*
2 ;8 6 mg#
200 irfLb
726^
;s 8 p
2 ;8 9
200
^78
7 3 ;3
;28o
2»8o
200
360
av erag e t o t a l Goneo o f e y s tin e in 200 ml;, o f s o l u t i o n
• $ T 2 mg«
370. mg;
’*26«=
T a b le s IV .A and IV B show 'th e p e rc e n ta g e o f tyrosin e, and
c y s tin e p a s s in g from, th e
solution=,
H o tio e t h a t a
g r e a t e r p e rc e n ta g e o f ty r o s in e p a s s e s th a n c y s t i n e ,
.
.
.-
.
T ab les V A
. .
■
and V B and T a b le s Vl A and Vl B5 showing th e p e rc e n t, of ty ro *
s in e and c y s tin e p a s s in g from th e HH^Ol and HaOl s o lu tio n s
r e s p e c tiv e ly ,, show th e same r e l a t i o n s h i p betw een th e amount- o f
ty r o s in e and c y s tin e p a s s in g ,
The p e rc e n ta g e o f th e two a c id s
.p a s sin g from th e HHjijOl s o lu t io n i s o n ly s l i g h t l y l a r g e r th a n
t h a t p a s s in g from th e HaOl s o lu t io n , b u t th e p e rc e n ta g e s from
b o th o f th e s e s o lu tio n s a r e c o n s id e r a b ly sm a lle r th a n th e p e r*
c e h ta g e s p a s s in g from th e
s o lu t io n ,
Hote th e r e f ­
l a t i o n s h ip betw een th e q u a n t ity o f e n c r u s ta ti o n and th e p e r*
e e h tag e o f c y s tin e and ty r o s in e p a s s in g .
The a v e ra g e w eight
•of th e (HHjij)g$0j^ -e n c r u s ta ti o n was £#«l |l grams and th e -p e r c e n t
o f ty r o s in e p a s s in g was 2,,.22, w h ile th e c y s tin e p a s s in g was
2 *08#*
w eighed
p a s s in g .
The average- e n c r u s ta ti o n fro m th e HHjijCJl .so lu tio n '
'
gram s, .w ith '1,0.76$'ty r o s in e and 1 ,6 6 ^ c y s tin e
The HaOl s o l u t i o n had ap av erag e e n c r u s ta ti o n o f
i{2,5 gram s, w ith 1 ,7 3 $ ty r o s in e and 1 ,5 8 $ c y s tin e p assin g ..
The c o n c e n tr a tio n o f c y s tin e and ty r o s in e p e r gram of e n ­
c r u s t a t i o n d id n o t fo llo w th e same o r d e r , however*
The con­
c e n t r a t i o n o f ty r o s in e p e r gram o f e n c r u s ta ti o n was h ig h e s t in
HaGl5 n e x t h ig h e s t i n (HH^)gSOjl and th e l e a s t i n HHj^Ol»
Sodium C h lo rid e c o n ta in e d th e h ig h e s t c o n c e n tr a tio n o f c y s tin e
. p e r g2?am# n e x t h ig h e s t i n CIIH^)^SO^5 and S l^ d l .co n tain ed th e
' le a s t's
' ■■■
•
•
•:
>»28«»
IV A
I3^ ro e n t T y ro sin e P a ssin g from th e H y d ro ly sa te
Q o n tain in g (HHi) gSG^
W eig h t. o f
Cone, o f T otal
S u rfac e
T y ro sin e T y ro sin e
C ru st , . H eading , p e r gram. P a ssin g
# 2*72 g*
83
,210 mg.
2 l»20
82
,,181
5 7 ,9 2
86
,1 6 9
5 5 , 81).
BI).
5 2 ,5 5
:
f r a c t i o n . . # P assin g
■
- :0^,40
11*064 mg-,' 1 1 .0 6 4
461*00*
2 ,0 2
9 .3 1 2
~ 45T
2 .1 2
9»772
9,773
46i
a l 98
1 1 ,0 6 4
-iSsrr
81t ,5
*19^
10,188
2 8 ,3 0
8 2 ,2
,1 7 6
1 0,261
10,261
""461"
2<23
5 9 ,1 0
86
.169
9,988
9.988
461
2 .1 7
TTBJ mg*
5 5 ,4 1 g .
1 1 .0 6 4
2,46
$
.
2.21
1 0 ,2 3 6 mg.
=
,420
Io n ie c o n c e n tr a tio n
-
»81j.O gram c a tio n
C o n c e n tra tio n o f ty r o s in e p e r 10*236
gram c a tio n o f (MHj^)^SO^
aoliO
-
12*18 mg®
Moles of
(IIV 2 S\
p a s s in g
2 2 , 4l
132
if- mg* o f ty r o s in e i n 200 m l. of. h y d ro ly s a te
2P
i? B
. P e rc e n t C ystine. P a ssin g Prom th e K y d n o ly sste
Go u t a in in g {
gSO^
W eight o f
Oono. of T o ta l
S u rfac e ■ ■i . • ■ C y stin e
■C y stin e
C r u s t"
Reading p e r gram P a s s in g .
F r a c tio n
^ P a ssin g
52,72 8*
89.5
fl^B mg#. 10^13
i. ■
5 li5 0
:.
80
.200
lo ,3 0
57.85
79.5
.SCR
'4
H #8o
11,80
31(1".
2,18
SSM
79
o210
11,73
' ',*•
11,73
3@ T .
2*17
' ■■■
■
,
52.55
79.5
58^30
79
59.1Q
80
.
g.
^ 30,43
%
1.87
"■ ■» "1 ,
1 ,9 k ■-
•,r-" ‘
#201}.
10,72
.
10,72
1I S T
1 . 98
. :
o210
12.2R
12.2k
2*2$
e,200
11.82
110 82
5%i
2,18
*' *
.203 mg.
M oles o f (BB^ ) gSCj,' p a s s in g
KoB1^
.
% »!{.20
8lj.C gram c a tio n .
I o n ic c o n c e n tr a tio n
C o n c e n tra tio n o f c y s tin e
■ l l 625'
p e r gram c a tio n o f (W ^ g S O ^ ToRa
13*^9 mg,
# mg-e of c y s tin e i n 200 mlV o f h y d r o ly s a te
TABLE V A
P e rc e n t T y ro sin e P a ssin g from th e H y d ro ly sa te
C o n ta in in g SH^Gl
W eight o f
S u rfac e
Reading
C ru st
Cone* o f
T y ro sin e
p e r gram
T o ta l
T y ro sin e
P a ssin g
8 ^ .g
.1 7 4
8*265 mg.
87
Jl6 0
6 ,9 6 0
I O60
k k -* 3Q
87
.160
'7 .1 3 4
1 ,6 4
4 7 .0 0
8 2 .5
*174
8,178
8^78
1^90
4 3 .8 0
85
,183
8 .0 0 4
8 .0 0 4
435
1*84
46«30
8 2 .3
.1 7 4
8.091
W
1 .8 6
4 4 .7 0
87
»160
7 .C 04
2^ k
1 .6 1
»169 mg.
7 ,6 6 2 mg,
4 5 .4 5 g ,
Moles o f HEjCl p a s eIng
3 3 ,5 0
s
Io n ic c o n c e n tr a tio n
C o n c e n tra tio n o f ' ty r o s in e
p e r gram c a tio n , of HH^Ol
# mgi o f ty r o s in e i n 200
F r a c tio n
'6 . 2 6 2
435 *
. % P a ssin g
1^90
X T fB #
,849
.849 gram c a tio n
1Tm g
’
9 .0 3 mg.
o f h y d ro ly s a te
TABD# V B
P e ro e n t Q y stin e P a ssin g from th e H y d ro ly sa te
Q o n tain in g
W eight of
S u rfa c e
C ru st
R eading
G"
80*2
Cone* o f
C y stin e
p e r gram
T o ta l
C y s tin e
P a ssin g
F r a c tio n
fa P a s s in g
6193 mg=
9.181 mg,
9.181
1 .7 7
8 .0 9 2
B lB Z
1*28
6 l* 2
• .182
8.0&2
8 1 ,2
.182
8.063
80
=200
U ,8 0
81
.181).
0*0#
&6.30
8 0 .2
.1 9 3
8*922
i|lio70
80*2
A 93
8.6 1 0
Z B p mg.
B Z Z mg*
W-k 30
"■ ■' .
g,
Holed o f HHj^Cl p a s s in g
M #
IiW ' "
W
1 0 ;
!,.sa:-;"'
1 .2 2
1 ,7 2
Iftf
-
Io n ic c o n c e n tr a tio n
C o n c e n tra tio n o f c y s tin e
p e r gram c a t i o n o f HH^Ql
1 * 2 2 .'/
1 .6 6
IZ B ^
.s to
08I4.9 gram c a tio n
6*621
" x "
1 0 ,1 $ mg.
# mg*, o f c y s tin e i n 200 ml* o f ,h y d ro ly s a te "
#AB&% 7$ A
Peyeent ty r o s in e Paealng from the H ydrolysate
G o n tain in g HaOl
: W eight o f
Gone* o f T o ta l
S a rfa e e .
T y ro sIne T y ro sin e
■G r a s t ■ H eading , p e r ' gram. P a ssin g . .
%3$ .g# ■ 8%
.SOlt mg.
8*82% mg*
ib W
*188
7 , 7%6
82
F r a e tio n . . ^ P a ssin g
1*03
lt6 0
W
%.$8
6 k . S-
W *7.
6k
*198
8*6#8
8.6 0 2
8*303
u ii
1*71
W
:
H oles o f HaGl p a ssin g
a
.786
I o.nie e o n e e n tr a tio n
s
o726 gram n a t io n
1 ^ # 'g*
*197 %$*
mg.
'i
G o n e e n tra tlo n o f ty r o s in e
p e r gram n a tio n o f JfaGl
8.376
* mg* o f ty r o s in e in 200 m l ,• of h y d r o ly s a te
11.2% ms*
r'
i.
P e v c e n t of: Oystiine P a ssin g :f2?om th e E y d ro ly satie
■ Gbatiaihimg SaOl
,:
Gone 0- of f e t a l
Gystiihe"- ■Oystiihe
p e r gram ^ P a s s i n g .. . . f r a c t i o n
w eig h t o f
- S n rfa c e
Grusti
Beading.
*8 X0 mg*
9 , 1 1 4 , mg*
$* 1 1 ^
^ M assing
1*60
270 a
. *.2 13
8 ,8 9 3
8*391
^70
1 .*# - - -
78
^ 228
9 , 9W
9 .9 4 6
Z? 0
1 ,7 4
80
»200
8 , 14:0
8+140
370"
1+43
i]lo 2
7 &*g
#*8
404?
-WNtihWir ■
IlB
IlITlI I' I'lll' fnh
11111)11^1
g,
»212 mg»
Moles o f SaOl p a s s in g
$6 013 -mg*.
, M s i '.
# 3 '
*
lo n io oonoentiratiion
O bnoentiratiion o f Gystiine
p ep gram c a tio n o f SstOl
* . . *726 -
9 o_013
. jf
* mgt of oystiihe In 200 ml*.of hydrolysate
0-726 gram n a t io n
1 2 o.i|l mg* ■
>
-Zh*, ,
: . - ,
PART, I I :
A numbetp o f c o n tr o l e x p e rim e n ts were ru n "as i n g . s e v e ra l ,. ^
d i f f e r e n t ..amino, a e l d s , ; amino, a e id s a l t S^ and In o rg a n ic s a l t s . in a n . e f f o r t to f in d some, o f tb d f a c t o r s g o v ern in g v p e r c r y s t a l ­
lig a tio n ,
.:.
'
;, ■ , .
A s o lu tio n c o n ta in in g one gram o f a la n in e , i n 5>0 m i. of
w a te r was p la c e d i n , a c e llo p h a n e c a s In g 0- , A, second such s o l ­
u t i o n a ls o c o n ta in in g (HB^)^SO^ was p re p a re d ,
c o n ta in in g one gram o f ly s in e - h y d r o c h lo r id e in
was p la c e d in a c e llo p h a n e h ag ,
A s o lu tio n
ml* of w ate r
O y s tin e -h y d ro c h lori.de was p r e ­
p a re d by d is s o lv in g c y s t i n e . i n th e minimum amount o f 6S HOl
w hich.w ould d is s o lv e , a l l o f. th e c y s t i n e and d i l u t i n g i t w ith
w ate r to 50 ml*
casin g *
T h is s o lu tio n , was p la c e d in a c e llo p h a n e
The sodium s a l t o f c y s tin e was p re p a re d b y adding th e
minimum amount o f 6 l HaOH w hich w ould d is s o lv e a Q u a n tity of
c y s tin e .
The s o lu t io n was d i l u t e d w ith w ater to 50 ml* and
p la c e d i n a c e llo p h a n e easing*.. A ll o f th e s e ’ c a s in g s w ere• hung
up f o r one week a t room te m p e ra tu re ,
R e s u lts
The volume i n ea ch c a s in g was red u ced to betw een 5 and 10
n£L, a f t e r one week.
The s o lu t io n c o n ta in in g p u re a la n in e
showed no p e r c r y s t a l l i s a t i o n ,
The c a sin g c o n ta in in g a la n in e
and (HHj^)2 SO^ had d ev elo p ed an e n c r u s ta tio n on th e s u r f a c e ,
T h is m a te r ia l gave a p o s i t i v e n in h y d rin t e s t , in d ic a t in g th e
p re se n c e o f an amino acid* . Ho e n c r u s ta tio n d ev elo p ed on th e
c a s in g s c o n ta in in g I ysine»hydrochlonidO j, <cy atin e ^ h y d n o o M o n id e,
op
th e sodium s a l t o f cystine.# - J n th e l a t t e r two, -th e c a s in g s
had le a k e d due t o th e w eakening o f th e membrane. b y a c id i n th e
ohe. and b ase i n th e o th er#
C y stin e i n th e P re sen c e o f In o rg a n ic S a l t s
F our s o lu t io n s c o n ta in in g #5 gram s c y s tin e i n J>0 ml.# o f ,
w a te r were p re p a re d by d is s o lv in g th e c y s tin e i n minimum quan­
t i t i e s o f 6 b HOl and th e n d i l u t i n g w ith w ater#
was added one gram o f ( M^ ) 2S
$o one s o lu t io n
t o a n o th e r one. gram o f
t o a n o th e r one gram o f HaGl5 to th e f o u r th was added two grams
o f (HH]^)gSOr.
A f i f t h s o lu tio n c o n ta in in g one gram o f c y s tin e
i n 50 ml# o f w a te r was p re p a re d and t o t h i s was added one gram
o f (BHi^)2 SO^9
These s o lu tio n s w ere p la c e d i n c e llo p h a n e
c a s in g s and l e f t h an g in g f o r th r e e days#
A ll th e c a s in g s h ad e n c r u s ta tio n s on th e s u r f a c e , b u t
S e v e ra l c a s in g s had leak ed #
lihen th e e n c r u s ta ti o n on th e o th e r
c a s in g s were removed, th e y a ls o broke#
The a c i d i t y . o f th e s o l*
u t io n s were such t h a t th e y weakened th e membranes' t o th e p o in t
t h a t th e y le a k e d o r b ro k e e a s ily #
were p o s s i b l e .
Thus no q u a n t i t a t i v e r e s u l t s
The e n c r u s ta ti o n s c o l l e c t e d , how ever, produced
p o s i t i v e n in h y d r in t e s t s #
A n o th er a tte m p t u s in g l e s s a c id t o
d is s o lv e th e c y s tin e r e s u l t e d i n th e same d i f f i c u l t y .
E x p erim en ts W ith C o llo d io n Membranes
■
. F iv e c o llo d io n membranes i n th e shape o f t e n in c h t e s t
tu b e s were made and f i l l e d w ith s o lu tio n s c o n ta in in g alan in e ,,
c y s tin e - h y d r o c h lo r id e ,' s o d iu m .s a lt o f c y s tin e , a p r o t e i n ..
h y d r o ly s a te c o n ta in in g
and a s o lu tio n c o n ta in in g
c y s t i n e , t y r o s i n e , and
r e s p e c tiv e ly *
These hags
were hung up and l e f t f o r one week a t room te m p e ra tu re « At
th e end o f t h a t p e r io d , no e n c r u s t a t i o n had d ev elo p ed on any
o f th e b a g s , and th e volume had. been re d u c e d v e ry l i t t l e * .
L arge b e a k e rs were placed, around th e bags and h e a t ap p lied ,
w ith bunsen b u r n e r s .
W ith h e a t a p p lie d f o r s e v e r a l, h o u rs,,
and th e te m p e ra tu re o f th e a i r around th e bags a s h ig h as
95^0,. no p e r o r y s t a l l i g a t i o n to o k p la c e *
Measurement o f th e Amounts o f In o rg a n ic S a l t s
Which P ass Through a C ello p h an e Membrane
I t i s n e c e s s a r y t o know th e r e l a t i v e ' amount o f an in ­
o rg a n ic s a l t w hich p a s s e s th ro u g h a membrane, i n o rd e r t o 'j u d g
th e e f f e c t t h a t s a l t h a s on th e amount of c y s tin e o r ty r o s in e
w hich w i l l p a s s th ro u g h th e membrane»
Two m olar s o lu tio n s of
M k Q l, BaGl, (BE, ) gSOi and a one m olar s o l u t i o n o f (
were p rep ared *
^SO^
Three 100 ml. q u a n t i t i e s o f each s o lu t io n were
p la c e d in c e llo p h a n e ca sin g s*
The amount o f e n c r u s ta ti o n on
ea ch c a s in g was w eighed and th e p e r c e n t p a s s in g th ro u g h was
c a lc u la te d ,"
T able V II shows th e r e s u l t s .
The s o l u b i l i t y i n
i
gram s p e r 100 grams o f w ater o f ea c h s a l t i s a ls o in d ic a te d .
. .
\ ' TABLE V II
.
P e rc e n t o f In o rg a n ic S a l t P a ssin g 'Through a
.,Oellophane Membrane
-. -
S a lt
m^oi
n
, : Gone„ i n ,
g/XOQ ml*
M o la rity
.BLO
o f -
2 m olar
if
ft
Ia C l
• t?
2 m olar
«
« ' ! '
«• "
ft
it
it
it
it
it .
gram c a tio n s p a s se d
if
1.366
12,67
1 .4 3 8
13*44
If
7 5 .4
8#
it
tt
gram c a tio n s p a sse d
• ,■/
12.62
1 3 ,0 4
1 .5 0 0
12*80
1 2 *82^
* .077 .
it
SO^ I m olar 13*2 g ,
it
it
m oles p a s se d © *037
"l4 *6 l,
'1*526
75+4 g«
it
gram c a tio n s p a sse d
.Ol^
M
i . 5 oos*
1 «4 ? 6g 0
W
2 m o lar 26,If. g*
. Tf
ft
m oles p a sse d a
g*
11*7 g*
3^o.o g*
" nv
• ’ . ' ' if '
m oles p a s s e d -H3 <>07 ?
( r a ^ , ?SOj^
Grams
. P asse d , $ P assed
Gram c a tio n s p a sse d rn a 080
.0 8 0
;t
$$
H 2 O at 2 0 0
IO 0T g o
3 7 d2
'
f?' '■ ' - 1 - ii'
ft
moles p a s se d »
Sol* .in
g/lOOg o f
a
5 +7 3 9 8 *
21*74
5 ,9 3 2
2 2 .4 7
6 .0 2 2
2 2 .8 1
,0 9 0
1 .5 8 1 s*
11*98
1.620
12,27
1*598
12,11
1 2 ,1 2 #
: .0 7 4
- .
.
'.
Bl&OtlS&IO#--.
,
' : ..
. ...
A&l&o a c id s a&d .Iaorganic aai&a a lik e would ab t pasa '
tlirough .a c o llo d io n membrane u n d e r th e c o n d itio n s ■d e s c rib e d i n
th e ex p e rim e n ta l. work» - I t i s c o n c e iv a b le t h a t i f i t had b een
p o s s ib le to make t h i n n e r ? more d e l i c a t e c o llo d io n membranes,
th a t, i n o r g a n i c . s a l t s , and p o s s i b ly amino a c id s a lo n g w ith th e
s a l t s s would have p a s se d th ro u g h th e membranes*
might conclude th a t Sober
Eowevsrs one
worked under d if f e r e n t ,Cpn-
.
d l t i o n s o r u sed c o llo d io n membranes w ith a d d i tio n a l components
When he su cceed ed I n p e r c r y s t a l l I S ing h i s t i d i n e from, an amino
a c id s o lu tio n *
,
Ih e f a c t t h a t n e i t h e r amino a c id s in s a l t f r e e s o lu tio n ,
n o r amino a c id s a l t s w ould p a ss th ro u g h a c e llo p h a n e membrane,
le a d s to th e c o n c lu s io n t h a t when sm all Q u a n titie s o f amino
a c id s p a ss through a membrane a lo n g w ith in o rg a n ic S a lta s= th ey
a re . m e re ly .c a r r ie d a lo n g ,I n . some way by th e s a l t *
T ables IV , Vs and Vl show t h a t th e
: >
s o lu tio n -
p a s se d a g r e a t e r p e rc e n ta g e o f c y s tin e and ty r o s in e th r o u g h .a '
c e llo p h a n e membrane th a n e i t h e r SfH^l o r I a S l 0
The (BE^)g 8
e n c r u s ta ti o n w eighed more th a n th e o th e r two. s a l t s , which were
n e a r ly equal in weight*.
The q u a n tity o f c y s tin e and ty r o sin e
i n •th e s o l u t i o n i s c o n s ta n t, th u s th e g r e a t e r th© 'am ount o f , ■
S a lt p a s s in g , th e g r e a t e r th e t o t a l c y s tin e and ty r o s in e
p assin g*
■
T his e x p la in s why (M , ) g80^ p assed th e g r e a te s t Qdan-
t i t y o f c y s tin e and ty r o s in e th ro u g h th e membrane®
~3 9 .*
!Bbo SaOl e n c r u s ta ti o n c o n ta in e d th e h ig h e s t c o n c e n tr a tio n
of ty r o s in e and c y s tin e p e r gram, o f e n c r u s ta ti o n fo llo w e d by
(1#^) gSO^ and SH^Gl r e s p e c tiv e ly ^ ■'When th e m olar co n c en t •
t r a t i o t i s a r e c a lc u la te d ;, i t i s found t h a t th e m olar cone on**
t r a t i o n o f (HH^) pSOr i s ro u g h ly o n e - h a lf t h a t o f th e e th e r two
s a l t s p a s s in g th ro u g h th e c e llo p h a n e membrane*
Ammonium sul*.
f a t e h a s tw ic e th e c a tio n c o n c e n tr a tio n o f
ev ers
and EaGlKbow^,
rBhus d o u b lin g th e m olar c o n c e n tr a tio n o f
a
c a tio n c o n c e n tr a tio n o f *8i|,0 i s o b ta in e d , w h ile th e c a tio n con*
c e n t r a t i o n o f IE^Gl rem ain s a t »81|.9 and th e c a t i o n cone an?
t r a t i o n o f HaGl rem ain s a t 07 26i
One would e x p e c t
and HH^Gl to prom ote th e p a ss­
age of h ig h e r c o n c e n tr a tio n s o f c y s tin e and ty r o s in e th r o u g h ■■
th e c e llo p h a n e membrane th a n HaGl, i f a c o r r e l a t i o n e x is te d
betw een t h i s phenomenon and I o n ic c o n c e n tr a tio n ,
We f in d , how*?
e v e r , t h a t (HE^)gSO^ c o n ta in s th e h i g h e s t . c o n c e n tr a tio n of
ty r o s in e and c y s tin e p e r gram c a t i o n fo llo w e d by HaGl and HE^-Ol
re s p e c tiv e ly = .
!BhuS no c o r r e l a t i o n seems t o e x i s t betw een con~
c e n t r a t i o n of c a tio n s ’p a s s in g th ro u g h the, membrane and th e con­
c e n t r a t i o n o f c y s tin e and ty r o s in e p a s s in g th r o u g h .th e membrane
w ith th e In o rg a n ic sa lt*
'
'
•
f a b le V lI I n d i c a t e s , a s do th e r e s u l t s i n f a b l e s IV, Vi*
and VI, t h a t . (HHj^gSO^ p a s s e s th ro u g h th e c e llo p h a n e membrane
I n g r e a t e r q u a n t i t i e s th a n do HEhOl o r HaGl*:
A c o rre la tio n is
e v id e n t when one com pares th e p e r c e n t and m oles o f s a l t p a s s in g .
w ith th e s o l u h i l i t y o f th e s a l t s
One th u s c o n c lu d e s t h a t th e
q u a n t i t y o f in o r g a n ic s a l t p e r c r y s t a lliz in g i s som ew hat'de­
p en d a n t upon th e s o l u b i l i t y ,
S e v e ra l mechanisms can foe o f f e r e d a s e x p la n a tio n f o r th e
p e r c r y s t a l I i z a t I on phenomenon,.: In o rg a n ic s a l t s i o n i s e in
s o lu tio n , and p a ss th ro u g h a membrane i n io n ic .f o r m w ith th e
w a te r a s i t .e v a p o ra te s th ro u g h th e l a t t i c e s t r u c t u r e o f th e
membrane,
The a n io n s and c a tio n s u n i t e on the- s u r f a c e o f th e
membrane- and form c r y s t a l s w hich b u ild up t o a c o n s id e ra b le
s ia e ,.
Some- o f th e amino a c id m o le cu les form z w itte r lo n s
u n it e w ith anions or catio n s* and p a ss through th e membrane
w ith them,
Z w itterlo n s are la r g e , and o n ly a few g e t through
th e sp a c e s i n th e l a t t i c e w hich i s composed of c e l l u l o s e u n i t s ,.
The number o f a w itte r lo n s p a ssin g through depends a ls o upon
th e number o f an ion s and c a tio n s p a ssin g th ro u g h ,
As water
evap orates through th e membrane* th e s o lu tio n becomes more eo n c e n tr a te d , reach in g a p o in t where the s a l t b eg in s t o c r y s ta l*
I i a e on th e i n s i d e ,
t i t t l e p e r c r y e ta l l i g a t i o n w ill occur from
t h i s p o in t on,'' Thus the q u a n tity o f p e r e r y s t a l l i z a t i o n depends
upon th e s o l u b i l i t y o f th e s a l t becau se p e r e r y s t a l l i z a t i o n w i l l
p r a c t i c a l l y c e ase when c r y s t a l l i z a t i o n b e g in s in sid e , the m m
b ra n e , and b ecau se p e r e r y s t a l l i z a t i o n depends upon th e number
o f io n s formed and p a s s e d th r o u g h , th e membrane,
Oellophane i s produced from c e llu lo s e by th e v is c o s e pro*
c e s-s*
The " v is c o s e ” , c e llu l o s e a a n th a te , i s f o rc e d th ro u g h
-lafin e
slo ts
g en era ted
w ith
in to
c e llu lo se .
B l- li g lu c o sid e
b a b ly lin k e d
P ig m a n
lo se
b a b ly
an a c id
m o le c u le s
are
der ” a a ls
The
stru ctu re
lin k a g e
togeth er
(9)
b ath p r o d u c in g
and
of
re­
r em a in s b a s i c a l l y
th e
th e
lin e a r
e x p la in s
th e
effect
in
th e
m ic e lla r l a t t i c e
fo rces,
th e
so lu b iliz in g
due
to
is
Ms i n c e
of
fo llo w s,
s a l t s p r o b a b ly
H
/
CONaT."
sh eets
c h a in s
sam e,
are pro­
by h ydrogen b ond s.
as
h e ld
th in
th e
a c id s
c e llu lo se
sa lts
on c e l l u ­
m o le c u le s
pro­
b y h y d r o g e n b o n d s and Van
a c tio n
a r e p la c e m e n t
and
of
stro n g
o f bonds
a c id s
such as
and
COHO b y
-4?-
MT h e
of
a c tio n
c h a in s
due
h y d r o ly tic
lo ss
of
of
a c id s
to
fib e r
sa lts
p assed
a m in o
a c id s
a c id
or
th e
le a d s
sa lts
a c c o m p a n ie d
th e
to
b y a d e g r a d a tio n
g lu c o sid ic
d im in ish e d
and
th e
ru p tu re
th e
c h a in s,
or
e x p e r im e n ta l
th rou gh
so lu tio n ,
one m ust
of
is
lin k a g e s.
c h a in le n g th
M ild
and
s tr e n g th .”
drogen bonds lin k in g
In
sa lts
h y d r o ly sis
c o n d itio n in g
Thus a c id s
lin k a g e .
and
th e
m em b r a n e
a m in o a c i d
in
c o n c lu d e
th e
th at
w ork
sa lts
absence
th e
of
la ttic e
by r e p la c in g
by h y d r o ly z in g
it
was fo u n d
fro m n e u tr a l
fa ile d
in o r g a n ic
g lu c o sid ic
th a t
in o r g a n ic
so lu tio n ,
to p e r c r y s ta lliz e
in o r g a n ic
sa lt
s a lts .
or
hy­
but
from
From t h i s
s p e c ific a lly
th e
c a tio n h a s more e f f e c t on r u p tu r in g th e l a t t i c e th a n th e acid#
I t I s more l i k e l y t h a t th e hydrogen bonds were d is r u p te d by
' /
th e c a tio n s th a n t h a t th e g lu e o s ld ie lin k a g e s s e r e Iiy d ro ly se d 6
B videnee i n s u p p o rt o f t h i s view i s th e, f a c t t h a t In o rg a n ic
S a lts d id n o t p a s s th ro u g h c o llo d io n membranes Q C o llo d io n i s
p y ro x y lin ^ a n i t r a t e d c e l l u l o s e 9 h a v in g NO. g ro u p s i n p la c e
of some o f th e OH groups#
C o llo d io n ' membranes a re more r e ­
s i s t a n t t o th e e f f e c t s o f a c id ic o r b a s ic s o lu t io n s th a n c e l l o ­
ph an e,
I f c a tio n s r u p tu r e hydrogen bonds in p a s s in g th ro u g h
a membrane* i t i s e a sy to e x p la in why i t would be d i f f i c u l t f o r
s a l t s t o p a s s th ro u g h c o llo d io n .
However* i f c a tio n s hydro-.,
ly s e g lu e o s ld ie lin k a g e s In p a s s in g th ro u g h th e membrane*, i t
i s d i f f i c u l t t o e x p la in why s a l t s d id n o t p a s s th ro u g h a c o l l o ­
d io n membrane.
C o n clu siv e e v id en ce t h a t th e m o le cu lar l a t t i c e o f t h e ,
c e llo p h a n e membrane was a f f e c t e d t o some d eg ree I s found i n th e
f a c t t h a t a f t e r an e n c r u s ta ti o n was removed from a m e m b ra n e '
t i n y beads o f liq u id ,f o r m e d on th e s u rfa c e of th e membrane,.
E v a p o ra tio n soon to o k p la c e and e n c r u s ta ti o n was n o te d ,.
P e r-,
c r y s t a l l i s a t i o n to o k p la c e much more slo w ly on new ly hung mem­
b ra n e s and no n o t! c a b le l i q u i d co u ld be o b serv ed on th e su rfa ce *
fh u s th e l i q u i d d r o p le ts came th ro u g h th e e n la rg e d sp aces i n •
th e membrane cau sed by th e r u p tu r e o f lin k a g e s i n th e l a t t i c e
by one o f th e mechanisms d e s c rib e d above,
A g r e a t e r p e rc e n ta g e o f ty r o s in e p a sse d from each o f th e
tb f e e s a l t s o lu t io n s th a n <3.1d cy stin e= .
D his i s c o n s is t a n t w ith
what we .know a b o u t th e two a c id s & T y ro sin e h a s a m o lecu lar
w eight o f 1 81 a s compared to c y s tin e w ith a m o le c u la r w eight
o f 2!j.0«
The ty r o s in e m o lecu le i s more compact th a n th e c y s­
t i n e m oleOUle0
T y ro sin e i s s o lu b le i n w ater t o th e e x te n t o f
o0lp, grams p e r 100 ml* o f w ater* ' C y stin e h a s a w ater s o l ­
u b i l i t y o f .0 0 6 grams p e r 100 mV o f w ate r a t 20* o*
T y ro sin e
h a s a h ig h e r i o n i s a t i o n c o n s ta n t w h eth er i t be from 1c l a s s i c a l ,
S w itte r io n 5, o r a c id ic fo rm u la tio n o f io n is a tio n *
Thus
io n is in g t o a g r e a t e r d eg ree and b e in g s m a lle r i n s i z e , th e
ty r o s in e m o lecu le p a s s e s th ro u g h th e membrane i n g r e a t e r
q u a n t ity th a n cystine®
Kober (?) s t a t e d t h a t c o l l o i d a l m a te r ia l w i l l n o t p a ss
th ro u g h a membrane*
There i s a p o s s i b i l i t y t h a t th e amino
a c id s i n th e p r o t e i n d ig e s t were i n p a r t in th e c o l l o i d a l
s ta te *
However9 th e f a c t t h a t some c y s tin e and ty r o s in e p a s s e d
from th e s a l t c o n ta in in g s o lu tio n s p ro v e s t h a t i t co u ld n o t
a l l be i n th e c o l l o i d a l sta te * .
T hat no p e r c r y s t a l l i z a t i o n
to o k p la c e from pure, c o m p le te ly s o lu b le amino a c id s o l­
u t i o n s , f u r t h e r p ro v e s t h a t .c o l l p i d a l i t y was n o t th e Source
o f d if f ic u lt y ^
Much work must be done b e fo re th e mechanism o f p e r c r y s t a l l i s a t i o n can be f u l l y u n d ersto o d *
Work sh o u ld be done on a l l
o f th e amino a c id s and o th e r o rg a n ic s o lid s*
I n th e f i e l d of
amino a c id s , th e work sh o u ld be d ir e c te d to w ard th e w ater
s o lu b l'6 '-onea -to a v o id tW u s e o f a e i d . oa?- base w hich w eakens1',
th e membranes and p re v e n ts work w ith in d iv id u a l- amino a c id s
and in o r g a n ic s a l t s w ith o u t co n tam in an ts*
Hore r a p id 'a n d
convenient- q u a n t i t a t i v e m ethods sh o u ld a ls o M sought*
Ad*,
d i t i o n a l work,, sh o u ld be done w ith c o llo d io n membranes, 'In an
e f f o r t t o d u p lic a te Z o b er5s work a n d ' p erh ap s u l t i m a t e l y a r r iv e
a t a u s e f u l , q u a n t i t a t i v e o r q u a l i t a t i v e s e p a r a tio n method f o r
amino a c id s * ■
‘■
■ - 11■
I9
P e r c r y a t a l l i z a t i o n i s the c r y s t a l l i z a t i o n o f - a s o lu te on
th e s u r fa c e o f a membranes a f t e r h a v in g p a s s in g th ro u g h th e
membrane, w ith the- e v a p o ra te d w a te r^ from t h e , s o lu t io n w ith*
in®
2»
,
Under th e c o n d itio n s d e s c rib e d i n t h i s s tu d y , no p er*
I
"
^
c r y s t a l l i z a t i o n to o k p la c e from any ty p e o f s o l u t i o n i n a -eol»
lo d io n membrane *
3*
'
Mo p e r c r y s t a l l i z a t i o n o c c u rre d on c e llo p h a n e membranes
c o n ta in in g in o rg a n ic s a l t - f r e e p r o t e i n d i g e s t Sa
I
|j.9
*
«
1
'■.
Ho p e r c r y s t a l l i z a t i o n o c c u rre d on c e llo p h a n e membranes
5
c o n ta in in g amino a c id s a l t s such a s ly s in e - h y d r o c h lo r id e ,
c y s tin e - h y d r o c h lo r id e , and th e ' sodium s a l t o f c y stin e ,'.
5»
M easurable q u a n t i t i e s o f c y s tin e and t y r o s i n e p a sse d
,
4•
- :^ ■
th ro u g h membranes c o n ta in in g p r o t e i n d ig e s ts and in o rg a n ic
6.
The g r e a t e r th e q u a n t ity of in o rg a n ic s a l t p a s sin g
th ro u g h th e membrane, th e h ig h e r th e p e rc e n ta g e of amino a c id
p a sse d th ro u g h th e membrane ^
The q u a n tity of in o rg a n ic s a l t
p a ssin g through th e membrane depends p rim a rily on the water
s o l u b i l i t y o f th e s a l t .
7«
A s l i g h t l y h ig h e r p e rc e n ta g e o f ty r o s in e p a s s e s th ro u g h
th e membrane th a n c y s t i n e 5 as i s e x p e c te d because ty r o s in e h a s
'
■
'■
.
a low er m o le c u la r w eig h t and a h ig h e r io n iz a t io n c o n sta n t*
.8»
,A lth o u g h , th e .ex a ct mechanism of p e r c r y s t a l l i z a t i o n i s u n ­
known i t i s b e lie v e d t o envoive th e r u p tu r e o f hydrogen bonds
i n th e l a t t i c e s t r u c t u r e of th e c e llo p h a n e membrane by an io n s
and c a tio n s o f the in o rg a n ic s a l t »
Z w itte rio n s p ro b a b ly
a s s o c ia te w ith th e a n io n s and c a t i o n s y and are c a r r i e d th ro u g h
th e membrane w ith them*
u n i t e to form c r y s t a l s .
On th e s u rfa c e th e a n io n s and c a tio n s
WjhS“
'
I.
'
LITBRATOR# OITED
B lock , R* J.., and B o llin g , D iana, TBB AMIBO ACID
COMPOSITION Of PROTEINS AND FOOD,
Thomas5 S p r in g fie ld ,' I l l i n o i s , -
p , 162, C harles C,
{ 1 9 l0 )
®
2 ,.
B lock, R. JY, and B o llin g , Diana, ib id . p . 66, (19^#)*
3»
F arber, L io n e l, SCIENCE, 82
I).,
I g # (193# ) .
C ortner, R. A ,, G ortner, R* A. I r . , and G ortner, #* A**. ,
OUTLINES OF BIOGBEMISTRY, 3rd e d it io n p* #0 John W iley
and Sons In c , (19#3)»
#,
Cnlmaraes (lei F ohseca5 Fanl o, REVIEW BRASIL # IM , Ijb;,
1 2 1 -7 , 195-200 (19)42)«■ ■
6.
Holmes, H. N ,, INTRODUCTORY COLLOID CHEMISTRY 3rd e d it io n ,
p« 1 8 John W iley and Sons I n c 6 New York (193)}-).
7 »,
Rober, P. A0, J* Am Oheme Soe J 9
8a
Mecham, D* Ka5 J® B io le Ohem I S i 6ij3 (1943) •
9,
Pigman5 Vf, W , and Ooepp5 BI, M, JTr., OREMISTRY OF 53932
CARBOHYDRATES p*- #43
9 % ”® (1 9 1 7 ).
Aoademio P r e s s , New York (19# ) *
■ .<r -
I■<
1 1 4815
v
AOKNOWLBmSMENT, .
The a u th o r ta k e s t h i s o p p o rtu n ity to e x p re s s h i s deep :
a p p r e c ia tio n to D r. B. L . Johnaon5. u n d er whose g u id an ce t h i s
work was done, and. to ' D r0 ,Lawrence O0 B inder and D r. K enneth
J» D oerin g f o r t h e i r h e lp i n th e w r itin g ‘of th e t h e s i s ,
Ap­
p r e c i a t i o n i s a ls o e x p re s se d ,to D r0 Edward W. A nackers Dr»
Hay. W o o d riffs . and Graeme B aker f o r t h e i r a s s is ta n c e ,;
MONTANA STATE UNIVERSITY LIBRARIES
762 1001 5429
%578
Ea93p
o o p .2
114815
I s jc to n , I/. j).
P e r c r y e t a l l i a & t i o n . o f c y s t i i Ie
and t y r o s i n e
z:
A tn e
S i f S p
c » p .
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