The isolation and purification of Brassica juncea myrosinase and a study of its glycoprotein nature
by Kenneth Nordahl Thompson
A thesis submitted to the Graduate Faculty in partial fulfillment of the requirements for the degree of
DOCTOR OF PHILOSOPHY in Chemistry
Montana State University
© Copyright by Kenneth Nordahl Thompson (1966)
Abstract:
Myrosinase of high purity was isolated from seeds of yellow Oriental mustard,. Brassica juncea, by the
following sequence of purification steps: (1) water extraction of the milled oil-free seeds, (2)
ammdnium sulfate precipitation (40-80% saturated fraction recovered), (3) ethanol precipitation (50%
fraction recovered), (4) step-wise elution from carboxymethylcellu-lose, and(5) gel filtration through
Sephadex G-200. The enzyme-preparation, appeared to contain thioglucosidase and sulfatase activity
when incubated with sinigrin solution at 3 7°C; however, separation of the two enzymes was not
accomplished. A 250-fold purification of thioglucosidase and a 115-fold purification of sulfatase were
obtained.
Myrosinase isolated in this work was estimated to have a molecular weight of 320,000 ± 15,000 by a
gel filtration technique-and contained 19.2% carbohydrate. Analyses by paper and gas-liquid
chromatography showed the carbohydrate to include residues of L-arabinose, L-fucose, D-xylose,
D-mannose, D-galactose, D-glucose,-D-glucosamine, and N-acetyl-neuram-inic acid. Amino acid
analysis revealed a high glutamic and aspartic acid content and a low percentage of sulfur-containing
amino acids.
Digestion of myrosinase with a Streptomyces griseus protease and subsequent fractionation by a series
of gel filtrations served to partially characterize the carbohydrate present. Major amounts of the
carbohydrate appeared to be present as an associated heteropolysaccharide with an approximate
molecular weight of 55,000. This polysaccharide contained L-arabinose, D-xylose, D-glucose, and
D-galactose. Carbohydrate moities with molecular weights of approximately 1000 were indicated as
present.. These contained D-xylose, L-fucose, D-mannose,. D-glucose, D-glucosamine , and
N-acetyl-neuraminic acid; however, specific assignment of sugars to a particular moiety, or moieties to
a particular enzyme, was not accomplished. The high carbohydrate content (85%) and the presence of
serine, threonine, aspartic and glutamic acids in this oligosaccharide mixture suggested
covalently-bound carbohydrate in myrosinase similar to that in known glycoproteins.
Treatment of myrosinase with 8 M urea solution followed by gel filtration appeared to free a large
polysaccharide from most protein present. Studies with urea supported the results obtained from the
protease digest.
THE ISOLATION AND PURIFICATION OF BRASSICA TUNCEA MYROSI NASE
AND A STUDY OF ITS GLYCOPROTEIN NATURE
by
KENNETH N (/TH O M PSO N
A t h e s i s s u b m itte d to th e G ra d u a te F a c u lty in p a r tia l
fu lfillm e n t o f th e re q u ire m e n ts fo r th e d e g re e
of
DOCTOR OF PHILOSOPHY
in
C h e m is try
A pproved:
H e a d , M a jo r D e p a rtm e n t
C h a irm a n , E x am in in g C o m m itte e
G ra d u a te D e a n
MONTANA STATE UNIVERSITY
B o zem an , M o n tan a
M a r c h , 1966
i ii
• ACKNOWLEDGEMENT
I w is h to e x p r e s s my s in c e r e g ra titu d e to Dr. K. J. G o e rin g for h is
g u id a n c e a n d c o n s id e r a tio n d u rin g my y e a rs o f g ra d u a te stu d y .
I w is h to a c k n o w le d g e th e f in a n c ia l a s s i s t a n c e p ro v id e d by th e
N a tio n a l S c ie n c e F o u n d a tio n r e s e a r c h g ra n t GB647 (a c o n tin u a tio n of
G 7 4 9 7 ), a n d b y th e M o n tan a . S ta te U n iv e rs ity C h e m is try D e p a rtm e n t an d
C h e m is try S ta tio n .
’
I w is h to th a n k th e M . S.U . C h e m is try D e p a rtm e n t s ta f f a n d g ra d u a te
s tu d e n ts for th e ir a d v ic e a n d a s s i s t a n c e .
I am in d e b te d to Dr. John R o b b in s o f th e U n iv e rs ity o f O re g o n fo r o b ­
ta in in g th e s e d im e n ta tio n p a tte r n s c o n ta in e d in th is t h e s i s .
And l a s t , b u t m o st o f a l l , I w is h to th a n k my p a re n ts for th e h e lp a n d
e n c o u ra g e m e n t w h ic h th e y h a v e g e n e r o u s ly g iv e n .
iv
TABLE OF CONTENTS
P ag e
LIST OF TABLES
LIST O F FIGURES
.................................... ... . .................... ................................................
..................................................... ...................................... ...
A B STR A C T.................................... ......................... ................................... v
v
vi
'...............v iii
IN T R O D U C T IO N ......................
I
MATERIALS AND METHODS
5
•A s s a y fo r M y ro sin a se - A c tiv ity
.......................... ... .................
5
P ro te in A s s a y s . ...................................................................................................................6
■C a rb o h y d ra te A s s a y .................................................................................................. ' 6
P re p a ra tio n o f I o p - E x c h a n g e C e l l u l o s e s ............. ..........................................
7
P r e p a r a tio n 'o f G el F iltr a tio n M e d ia ..................................................................
7
E le c tro p h o re s is P ro c e d u re s
.......................................... ... . . .
8
• P a p e r C h ro m a to g ra p h y T e c h n iq u e s ..................................................................
10
- Amino A cid A n a ly s is ...................................................................................................... 10
• S e d im e n ta tio n S tu d ie s . ....................... ......................................................... ... .
11
G a s - L iq u id C h ro m a to g ra p h y o f C a r b o h y d r a t e s ............. ...................... ... .
11
EXPERIMENTAL.....................................................................•..................
■E x tra c tio n a n d P u r ific a tio n o f M y ro s in a s e . . . . . . . . . . . . . . . .
C h e m ic a l C o m p o sitio n o f M y ro s in a s e . . . . . i . . . . . . . . . . . .
M o le c u la r W e ig h t o f M y ro s in a s e ................................
■P ro n a s e -D ig e s tio n o f M y ro s in a s e ..................................................................
■D is s o c ia tio n o f M y ro s in a s e .................' .................... ......................... ■. . .
14
..
.
.
.
D IS C U S S IO N ........................................................................
. E x tra c tio n a n d .P u rif ic a tio n o f M y r o s i n a s e ............. ■............................... .
C h e m ic a l C o m p o sitio n o f M y r o s i n a s e ..........................................
M o le c u la r W e ig h t of M y ro s in a s e . . ......................................... - ...................
P ro n a s e a n d - D is s o c ia tio n - S tu d ie s o n M y r o s i n a s e ....................................
14,
20
32
35
49
53
53
56
58
59
SU M M A R Y ................................
64
LITERATURE CITED . .
65
V
I
LIST OF TABLES
TABLE
•
'
P ag e
I „ P u r ific a tio n o f-B ra s s ic a ju n c e a M y ro s in a s e
I I . C h e m ic a l C o m p o sitio n o f M y ro s in a s e
I I I . - Amino A cid C o m p o sitio n o f M y ro s in a s e
........................................... ... . 21
. . . . . . . . . . . . . . . . . . . .
. . ....................... ...
22
24
IV. C o m p o sitio n o f th e -C a r b o h y d ra te - A s s o c ia te d w ith
M y r o s i n a s e ............. ..............................................................................................
28
■V. C a rb o h y d ra te C o m p o s itio n o f th e P r o n a s e - M y r o s in a s e
D ig e s t F r a c tio n s ........................................ .............................................................
44
V I. Amino A cid C o m p o sitio n o f th e P r o n a s e - M y r o s in a s e
D ig e s t F r a c tio n s
..................................................................................................48
vi
LIST OF FIGURES
FIGURE
P ag e
1 . F r a c tio n a tio n o f c ru d e m y ro s in a s e b y s te p - w is e
e lu tio n from C M - c e llu lo s e ..................................................................
16
2 . S e d im e n ta tio n p a tte r n s o f m y ro s in a s e p re p a ra tio n s . . . . . . . . . . . .
17
3 . G el f iltr a tio n o f C M C -2 o n S e p h a d e x G -2 0 0 ....................... ...................... ... 18
4 . E lu tio n o f p u rifie d m y ro s in a s e from D E A E -ce llu lo se
w ith 0.05 M sodium c itr a te b u ffer, pH 6.1 ....................... ...
19
5 . D isk e le c tr o p h o r e s is o f p u rifie d m y ro s in a s e o n
p o ly a c ry la m id e g e l a t pH 8.3 . . . . . . . . . . . . . . . . . . . . . . . . . .
20
6 . G a s ch ro m a to g ra m o f th e tr im e th y ls ily l d e r iv a tiv e s
from th e 2 4 - h o u r m y ro s in a s e h y d ro ly z a te ........................................
25
7 . G a s ch ro m a to g ra m o f th e tr im e th y ls ily l d e r iv a tiv e s
from th e 4 8 -h o u r m y ro s in a s e h y d r o l y z a t e ............. ...
26
8 . G as ch ro m a to g ra m o f th e tr im e th y ls ily l d e r iv a tiv e s
from th e 72- h o u r m y ro s in a s e h y d ro ly z a te . . . . . . . . . . . . . . . . . .
27
9 . Rb d v s . m o le c u la r w e ig h t for Bio G e l P -3 0 0 (41x2.5 cm)
e q u ilib r a te d w ith 0.05 M sodium c itr a te b u ffer, pH 6.1 . . . . . . . . .
. 34
1 0 . G e l f iltr a tio n o f th e p r o n a s e - m y r o s in a s e d ig e s t o n
■S ep h a d eix
2 5 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
37
1 1 . G el f iltr a tio n o f th e p ro n a s e b la n k o n S e p h a d e x G -2 5 .................... . . . 38
1 2 . G e l f iltr a tio n o f P M -2 5-1 o n S e p h a d e x G - 75 . . . . . . . . . . . . . . . .
39
1 3 . G e l f iltr a tio n o f P -2 5 -1 o n S e p h a d e x G - 75 . . . . . . . . . . . . . . . . .
40
1 4 . G e l f iltr a tio n o f P - 2 5 -2 o n S e p h a d e x G - 75 ........................................................ 40
1 5 . G e l f iltr a tio n o f P M -2 5 -2 o n S e p h a d e x G -7 5 . . . . .
.......................
41
v ii
1 6 . G e l f iltr a tio n o f P M - 7 5 -2 ,(A) a n d P M - 7 5 -3 (B) on
-S e p h a d e x G -15 ....................... ...
42
1 7 . - Schem e fo r fr a c tio n a tio n b y g e l f iltr a tio n o f th e
c a rb o h y d ra te f r a c tio n s in th e p ro n a s e - m y r o s in a se
d ig e s t o r in th e prona s e b la n k ..................................................'............... ...
43
1 8 . G a s ch ro m a to g ra m o f t h e .tr im e th y ls ily l d e r iv a tiv e s
from th e 2 4 - h our P M - 75-1 h y d r o l y z a t e ............. ................................................45
1 9 . G a s ch ro m a to g ra m o f th e tr im e th y ls ily l d e r iv a tiv e s
from th e 2 4 - h o u r P M -15-1 h y d r o l y z a t e ........................................................... . 46
2 0 . G a s ch ro m a to g ra m o f th e tr im e th y ls ily l d e riv a tiv e s
from the- 2 4 - h o u r P M -1 5 -2 h y d r o l y z a t e .............................................................. 47
2 1 . G el f iltr a tio n o f a n 8 M u re a s o lu tio n o f m y ro s in a s e
on. S e p h a d e x G -2 0 0 (5 8 x 1.6 c m ) .................................... .................................... 50
22 . G e l f iltr a tio n o f a n 8 M u re a s o lu tio n o f m y ro s in a s e
on S e p h a d e x G -2 0 0 (4 7 x 1.6 c m ) .................................................
52
v iii
ABSTRACT
' M y ro s in a s e o f h ig h p u rity w a s i s o l a t e d from s e e d s o f y e llo w O rie n ta l
m u s ta rd ,. B ra s s ic a j u n c e a , b y th e fo llo w in g s e q u e n c e o f p u rif ic a tio n s te p s :
(I) w a te r e x tr a c tio n o f th e m ille d o i l - f r e e s e e d s , (2) am m dnium s u lf a te p re ­
c ip ita tio n - (40-80% s a tu r a te d fr a c tio n r e c o v e r e d ) , (3) e th a n o l p r e c ip ita tio n
(50% fr a c tio n r e c o v e r e d ) , (4) s te p - w is e e lu tio n from c a rb o x y m e th y lc e llu I o s e a n d (5) g e l f iltr a tio n th ro u g h S e p h a d e x G -2 0 0 . The e n z y m e -p re p a ra ­
tio n , a p p e a re d to c o n ta in th io g lu c o s id a s e an d s u lf a ta s e a c ti v ity w h en in c u ­
b a te d w ith s in ig rin s o lu tio n , a t 3 7 ° C ; h o w ev er, s e p a r a tio n o f th e tw o e n ­
z y m es w a s n o t a c c o m p lis h e d . A 2 5 0 -f o ld p u rif ic a tio n o f th io g lu c o s id a s e
a n d .a 1 1 5 -fo ld p u r if ic a tio n o f s u lf a ta s e w e re o b ta in e d .
M y ro s in a s e is o la te d in th is w ork w a s e s tim a te d to h a v e a m o le c u la r
w e ig h t o f 320,00-0 + 15,000 by a g e l f iltr a tio n te c h n iq u e -a n d c o n ta in e d 19.2%
c a r b o h y d r a te . A n a ly s e s b y p a p e r a n d g a s - l i q u i d c h ro m a to g ra p h y sh o w e d th e
c a rb o h y d ra te to in c lu d e - r e s id u e s o f L - a r a b i n o s e , L - f u c o s e , D - x y l o s e , D m a n n o se ,- D - g a l a c t o s e ,- D - g l u c o s e ,- D - g lu c o s a m in e , an d N - a c e ty I rn e u ra m in ic a c i d . Amino a c id a n a ly s is re v e a le d a h ig h g lu ta m ic a n d a s p a r tic a c id
c o n te n t a n d a lo w p e rc e n ta g e o f s u lf u r - c o n ta in in g am ino a c i d s .
D ig e s tio n o f m y r o s in a s e w ith a . S tre p to m y c e s g r is e u s p r o te a s e - a n d 1
s u b s e q u e n t fr a c tio n a tio n b y a s e r ie s o f g e l f iltr a tio n s s e r v e d to p a r tia lly
c h a r a c te r iz e th e c a rb o h y d ra te p r e s e n t . M a jo r a m o u n ts o f th e c a rb o h y d ra te
a p p e a re d to b e p r e s e n t a s a n a s s o c i a t e d h e te r o p o ly s a c c h a r id e w ith a n a p ­
p ro x im a te m o le c u la r w e ig h t o f 55,000. T h is p o ly s a c c h a r id e c o n ta in e d La r a b i , n o s e D - x y l o s e ,- D - g lu c o s e , a n d D - g a l a c t o s e . C a rb o h y d ra te m o itie s
w ith m o le c u la r w e ig h ts o f a p p ro x im a te ly 1000 w e re in d ic a te d a s p r e s e n t . .
T h e s e c o n ta in e d D - x y lo s e , L - f u c o s e , D - m annose,.- D - g lu c o s e , D - g lu c o s ­
a m in e , an d N - a c e ty l- n e u r a m in ic a c id ; h o w e v er, s p e c if ic a s s ig n m e n t of
s u g a r s to a p a r tic u la r m o iety , or m o ie tie s to a p a r tic u la r enzym e, w as n o t a c ­
c o m p lis h e d . The h ig h c a rb o h y d ra te c o n te n t (85%) a n d th e p r e s e n c e o f s e r ­
i n e , t h r e o n in e ,. a s p a r tic a n d .g lu tam ic a c id s in th is o lig o s a c c h a r id e m ix tu re
s u g g e s te d c o v a le n tly -b o u n d c a rb o h y d ra te in m y ro s in a s e s im ila r to th a t in
k n ow n g l y c o p r o te in s .
T re a tm e n t o f m y ro s in a s e w ith 8 M u re a s o lu tio n fo llo w e d b y g e l f i l t r a ­
tio n a p p e a re d to fre e a la rg e p o ly s a c c h a r id e from m o st p ro te in p r e s e n t.
S tu d ie s w ith u re a s u p p o rte d th e r e s u lts o b ta in e d from th e p r o te a s e d i g e s t .
INTRODUCTION
M y ro s in a s e is th e e n zy m e s y s te m p r e s e n t in s e v e r a l m em bers of
C r u c if e r a e , T r o p a e o la c e a e , C a p p a rid a c e a e a n d R e s e d a c e a e w h ic h is r e ­
s p o n s ib le for th e b re a k d o w n o f m u s ta rd o il g lu c o s id e s (I) in to g lu c o s e , i n ­
o rg a n ic s u lf a te a n d o rg a n ic is o th io c y a n a te (G m elin an d V irta n e n , 1959).
A llyl is o th io c y a n a te c a n b e is o la te d from m u sta rd s e e d , b u t o b s e r v a ­
tio n s o v e r a c e n tu ry ag o o n b la c k m u s ta rd re v e a le d th a t th is v o la tile m u s­
ta rd o il d id n o t o c c u r fre e in th e in ta c t s e e d (B o u tro n -C h a rla n d a n d R o b iq u e t, 1831; F a u r e , 1 8 3 1 ). T h e s e w o rk e rs sh o w e d th a t is o th io c y a n a te w as
lib e r a te d o n ly w hen c ru s h e d s e e d s w ere s u s p e n d e d in w a te r. In a d d itio n ,
th e y o b s e r v e d th a t if th e c ru s h e d s e e d s w e re p re tre a te d w ith p ro te in ­
d e n a tu rin g r e a g e n t s , m u s ta rd o il lib e r a tio n w a s p re v e n te d in th e s u b s e q u e n t
w a te r s u s p e n s i o n .
A g lu c o s id e is o la te d from b la c k m u s ta rd s e e d p ro v id e d e v id e n c e th a t
g lu c o s e a n d in o rg a n ic s u lf a te w ere a s s o c i a t e d w ith th e is o th io c y a n a te in
th e s e e d (B ussy, 1 8 4 0 ). B u ssy d is c o v e re d th a t a m ix tu re o f th is g lu c o s id e
,2
■a n d a w a te r - s o lu b le p ro te in e x tr a c t from m u s ta rd s e e d c a u s e d th e a p p e a r­
a n c e o f g l u c o s e , in o rg a n ic s u lf a te an d a Ily l i s o t h i o c y a n a t e . The g lu c o s id e
w a s n am ed a s th e s a l t o f m y ro n ic a c id a n d t h i s la te r le d to n am in g i t s h y ­
d ro ly s in g e n z y m e , m y ro s in . T o d ay th is g lu c o s id e is k now n a s s in ig rin (I,
w h e re R = C H 2 = C H -C H 2- . As a r e s u lt, th e e n zy m e s y s te m h a s b e e n v a rio u s ly
re fe rre d to a s m y ro s in , m y ro s in a s e o r s in ig r in a s e .
I n it ia l s tu d ie s o n m y ro s in a s e s u g g e s te d th e p r e s e n c e o f tw o e n z y m e s ,
a th io g lu c o s id a s e a n d a s u lf a ta s e (von E u ler a n d E ric k s o n , 1926). S an d b erg
a n d H o lly (1932) c o n firm e d t h e s e r e s u l t s a n d , s h o rtly th e r e a f te r ,, a c tu a l
s e p a r a tio n o f th io g lu c o s id a s e a c ti v ity from s u l f a t a s'e a c ti v ity w a s re p o rte d
(N eu b erg a n d S c h o e n e b e c k , 1 9 3 3 ).
• A fter tw o d e c a d e s o f a p p a re n t a c c e p t a n c e , th e tw o -e n z y m e c h a r a c te r
o f m y ro s in a s e w a s q u e s tio n e d (E ttlin g e r a n d L u n d e e n , 1956; 1957). O ne en­
zym e , a t h i o g lu c o s id a s e , w a s p o s tu la te d to a c t fo llo w e d b y a s ta b iliz in g
n o n -e n z y m a tic re a rra n g e m e n t th a t lib e r a te d s u lf a te a n d fo rm ed a Ily l is o th io ­
c y a n a te . E x p e rim e n ta l d a ta a p p e a re d la te r to su p p o rt th is p re m is e (N a g a sh im a a n d U c h iy a m a , 1 9 5 9 a -d ).
N ew e v id e n c e fo r a tw o -e n z y m e s y s te m w a s o b ta in e d w h en m e th o d s
d iff e r e n t from th o s e o f N e u b erg an d S c h o e n e b e c k a llo w e d s e p a r a tio n o f th e
tw o a p p a r e n t en zy m e a c t i v i t i e s in y e llo w O rie n ta l m u s ta rd s e e d ,. B ra s s ic a
J u n c e a (G a in e s a n d G o e rin g , 1960; G a in e s , 1960). W h en in c u b a te d in d i­
v id u a lly w ith s in ig r in , t h e s e en zy m es r e l e a s e d o n ly o n e o f th e c h a r a c te r is ­
t i c p r o d u c ts , g lu c o s e or in o rg a n ic s u lf a te ; h o w ev er, if th e y w e re m ix ed to ­
g e th e r p rio r to in c u b a tio n c o m p le te h y d r o ly s is o f s in ig rin w a s a c h ie v e d .
3
E a c h e n zy m e w a s shovyn to p o s s e s s h y d ro ly tic a c ti v ity to w a rd s e v e r a l s y n ­
th e t i c s u b s tr a te s ( G a in e s , 1960; G a in e s a n d G o e rin g , 1960; 1962).
T he d is a g r e e m e n t b e tw e e n th e o n e -e n z y m e an d th e tw o -e n z y m e
th e o r ie s is s t i l l n o t s e t t l e d .
The th io g lu c o s id a s e p re p a ra tio n o f G a in e s a n d .G o e rin g (1962) b e h a v e d
g e n e r a lly a s a /5 - g lu c o s id a s e a n d in com m on w ith sorpe o th e r /S - g lu c o s id a s e s (H e lfe ric h , 1943) w a s sh o w n to c o n ta in c a rb o h y d ra te ( G a in e s , 1960).
M an y g ly c o s i d a s e s a re a s s o c i a t e d w ith p o ly s a c c h a r id e s w h ic h see m to
s t a b i l i z e t h e s e s y s te m s . If for som e r e a s o n th i s c a rb o h y d ra te is e lim in a te d
d u rin g p u r if ic a tio n fre q u e n tly th e en zy m e s y s te m b e c o m e s u n s t a b l e . In m o st
c a s e s i t is n o t k now n how t h e s e p o ly s a c c h a r id e s a re a tta c h e d to th e p ro te in
m o ie ty a n d i t is c o n c e iv a b le th a t m an y o f t h e s e s y s te m s a re m ix tu re s .o f
p o ly s a c c h a r id e s an d p ro te in ra th e r th a n , s p e c if ic m o le c u la r e n t i t i e s (F ish e r
an d S te in , 1960).
H u o ta ri (1962) o b ta in e d e v id e n c e fo r c o v a le n tly -b o n d e d c a rb o h y d ra te
in th e th io g lu c o s id a s e from y e llo w O rie n ta l m u s ta rd s e e d . T h is c a rb o h y ­
d ra te a p p e a re d to b e p r e s e n t a s a n o lig o s a c c h a r id e w h ic h w a s fre e o f s i a l i c
a c id a n d a c c o u n te d for o n e p e rc e n t o f th e th io g lu c o s id a s e p r e p a r a tio n . In
th e sa m e w o rk , g a l a c t o s e , a r a b i n o s e , h e x o s a m in e an d u ro n ic a c id w ere r e ­
p o rte d to b e p r e s e n t in th e c a rb o h y d ra te m o ie ty .
P ro te in s c o n ta in in g tig h tly -b o u n d c a rb o h y d ra te a re n u m e ro u s an d h a v e
b e e n know n fo r m any y e a r s . T hey a re c o n s ti tu e n ts o f b lo o d p la sm a , m u co u s,
c e l l w a ll m a te r ia l, c o n n e c tiv e t i s s u e a n d in c lu d e som e h o rm o n e s , b lo o d
g ro u p s u b s t a n c e s , a n tib o d ie s , a s w e ll a s e n z y m e s . S in c e t h e s e c o m p o u n d s
a re p a rt o f m any v ita l p r o c e s s e s in liv in g t h i n g s , th e ir s tr u c tu r e an d
4
m e ta b o lis m a re o f i n t e r e s t . E n zy m es c o n ta in in g c a rb o h y d ra te a re o f a d d e d
i n t e r e s t s in c e th e ir c a rb o h y d ra te m o ie tie s m ay s e rv e in th e e n z y m e s ' a c tiv e
s i t e s . ' H e lfe ric h e t alo (1938) s u g g e s te d th a t th e c a rb o h y d ra te g ro u p in g o f
j3-
g lu c o s i d a s e s m ay a c t a s a " s e e d in g " s ite n e c e s s a r y for e n zy m e a c tiv ity .
H o w ev er, p re lim in a ry s tu d ie s in v o lv in g a m ild p e rio d a te tre a tm e n t o f c a rb o ­
h y d ra te -c o n ta in in g e n z y m e s s e e m e d to in d ic a te th a t th e c a rb o h y d ra te w a s
n o t e s s e n t i a l for en zy m e a c ti v ity (P azurert a L , 1963).
P rio r to 1950, s tu d ie s o n p ro te in s c o n ta in in g c a rb o h y d ra te w e re p ri­
m a rily d e s c r ip tiv e a n d h a v e b e e n a d e q u a te ly re v ie w e d (L e v e n e , 1926;
M ey er, 1945; S ta c e y , 1946; B e tte lh e im - J e v o n s , 1958). T h e s e a u th o rs p o in t
o u t th e c o n fu s e d n o m e n c la tu re w h ic h e x i s t s in th is f i e l d . S chm id (1964) h a s
u s e d th e term " g ly c o p ro te in s " to d e s c r ib e " . . . p r o te in s , e x c lu d in g n u c le o p r o te in s , th a t c a rry c o v a le n tly - b o u n d c a rb o h y d ra te " . N um erous g ly c o p ro te ­
in s h a v e b e e n d is c o v e r e d , b u t o n ly th re e o f th e s e are k n o w n to h a v e en zy m e
a c tiv ity . T h ey a re T a k a -a m y la s e A (T su g ita an d A k a b o ri, 1959), a c id d e o x y ­
r ib o n u c le a s e (B ernardi e t a L , 1965) a n d g lu c o s e o x id a s e (P azu r e t a l .,1965).
The s ig n if ic a n t am o u n t o f c a rb o h y d ra te th a t is a p p a re n tly lin k e d c o v a le n tly
in th e en zy m e m y ro s in a s e s u g g e s ts th a t th is s y s te m m ig h t be c l a s s i f i e d a s
a g ly c o p ro te in .
The p u rp o s e o f th is r e s e a r c h w a s to fu rth e r in v e s tig a te th e tw o en zy m e c h a r a c te r o f m y ro s in a s e an d th e n a tu re o f th e c a r b o h y d ra te -p r o te in
c o m p le x w h ic h i s p r e s e n t .
MATERIALS AND METHODS
A s s a y fo r M y ro s in a s e A c tiv ity
E n z y m a tic h y d r o ly s is o f s in ig rin b y m y ro s in a s e lib e r a te s g l u c o s e , in ­
o rg a n ic s u lf a te a n d a Ily l is o th io c y a n a te . E nzym e a s s a y s w e re m ade o n in ­
c u b a tio n m ix tu re s p re p a re d b y w arm ing 0.2 m l o f m y ro s in a s e s o lu tio n w ith
0.5 ml o f s in ig r in s o lu tio n (20 mg p e r ml) a t 3 7 ° C . , S o lu tio n s w e re in 0.05 M
so d iu m c itr a te b u ffer, pH 6.1. In c u b a tio n tim e s w ere v a rie d to p re v e n t to ta l
h y d ro ly s is o f s u b s t r a t e . The r e a c tio n w a s s to p p e d w ith d in itr o s a lic y lic a c id
s o lu tio n w h en a s s a y in g th io g lu c o s id a s e a c ti v ity an d w ith tr ic h lo r o a c e tic
a c id s o lu tio n w h en a s s a y in g s u lf a ta s e a c tiv ity .
S in ig rin w a s o b ta in e d from M an n R e s e a rc h L a b o r a to r ie s , C a lifo rn ia
C o rp o ra tio n fo r B io c h e m ic a l R e s e a r c h , a n d A ld ric h C h e m ic a l C om pany, I n c .
E a c h s in ig r in p re p a ra tio n w a s c h e c k e d fo r p u rity b y p a p e r c h ro m a to g ra p h y ,
a lo n g w ith a b la n k r e a c tio n c o n ta in in g a s s a y r e a g e n t s .
G lu c o s e lib e r a te d b y th e w a te r e n zy m e e x tr a c t w a s d e te rm in e d by
q u a n tita tiv e p a p e r c h ro m a to g ra p h y (W h is tle r a n d B eM iller, 19 62) u s in g n-'
b u ta n o l- p y r id in e - w a te r (6 :4 :3 , v /v ) s o lv e n t a n d th e p h e n o l- s u lf u r ic a c id
c o lo rim e tric p r o c e d u r e . The d in itr o s a lic y lic a c id m eth o d (Summer, 1925) w a s
u s e d to a s s a y g lu c o s e a t th e o th e r s ta g e s o f en zy m e p u r if ic a tio n . S u lfa te
w a s a n a ly z e d tu r b id im e tr ic a lly a s b ariu m s u lf a te (D o d g so n , 1961). A llyl
is o th io c y a n a te w a s n o t d e te r m in e d , a lth o u g h i t s o d o r w a s e v id e n t du rin g
e v e ry in c u b a tio n in w h ic h g lu c o s e a n d s u lf a te w ere l i b e r a te d .
S p e c if ic a c t i v i t y o f m y r o s in a s e w a s c a l c u l a t e d a s p r e v i o u s l y d o n e b y
G a in e s a n d G o e rin g (1962), w h e r e in a u n it o f en zy m e a c t i v i t y w a s d e fin e d
6
a s th a t am o u n t o f en zy m e w h ic h c a ta ly z e d th e h y d ro ly s is o f 5 mg o f s u b ­
s tr a te p e r h o u r a t 37°C .
P ro te in A s s a y s
Q u a n tita tiv e a n a ly s is fo r p ro te in (Lowry et- a L 1951) w as. a c c o m ■
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■ ■:
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:
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V
p lis h e d u s in g p h e n o l re a g e n t o b ta in e d from th e H a rtm a n -L e d d o n C om pany,
P h il a d e lp h ia , w ith eg g a lb u m in (C a lifo rn ia C o rp o ra tio n fo r B io c h e m ic a l Re­
s e a r c h , B G r a d e , 5X r e c r y s ta lliz e d ) a s th e s ta n d a r d . In d ilu te s o lu tio n
« 0 .1 0 mg p ro te in /m l), K a lc k a r's . (1947) a p p ro x im a tio n p ro v id e d a c o n v e n ie n t
a n d a d e q u a te p ro te in d e te r m in a tio n .
P ro te in e lu te d from v a rio u s c h ro m a to g ra p h ic c o lu m n s w a s fo llo w e d by
b y o p tic a l d e n s ity m e a s u re m e n ts a t 280 mji in e ith e r a B eckm an M o d el DB o r
M o d el DJC-2- s p e c tro p h o to m e te r.
C a rb o h y d ra te A s s a y
The p h e n o l- s u lf u r ic a c id m eth o d o f D u b o is e t a l . (1956) w a s u s e d for
q u a n tita tiv e a n a ly s is o f c a rb o h y d ra te in m y r o s in a s e . T h is m eth o d a llo w e d
th e d e te rm in a tio n o f c a rb o h y d ra te w ith o u t in te rfe re n c e from p ro te in (Keen
a n d O p i e , 1957). A c o m p o s ite s u g a r m ix tu re c o n ta in in g g a l a c t o s e , m a n n o s e ,
a r a b i n o s e , x y l o s e , g l u c o s e , fu c o s e a n d g lu c o s a m in e w a s u s e d .to p re p a re a
s ta n d a r d iz a tio n c u rv e fo r th is te c h n iq u e . The r e a c tio n m ix tu re w a s p re p a re d
b y m ix in g I m l of s o lu tio n c o n ta in in g c a rb o h y d ra te (10-100 jug) w ith I ml o f
5% r e d i s t i l l e d p h e n o l a n d th e n ra p id ly a d d in g 5 ml o f 95.0-98.0% s u lfu ric
a c i d . A fter 10 m in u te s th e s o lu tio n w a s c o o le d fo r 30 m in u te s in ru n n in g
ta p w a te r. The o p tic a l d e n s ity o f th e a m b e r-c o lo re d s o lu tio n w a s re a d a t
'
7
490
o n a B eckm an M o d el DB s p e c tro p h o to m e te r. C a rb o h y d ra te e lu te d
d u rin g co lu m n c h ro m a to g ra p h y w a s fo llo w e d by th e sam e m e th o d .
P re p a ra tio n o f Io n -E x c h a n g e C e llu lo s e s
D ie th y la m in o e th y lc e llu lo s e (D E A E -cellu lo se) o b ta in e d from E astm an
O rg a n ic C h e m ic a ls a n d c a r b o x y m e th y lc e llu lo s e (C M -c e llu lo s e ) o b ta in e d
from Sigm a C h e m ic a l C o m p an y w e re b o th p re p a re d fo r u s e in th e re c o m ­
m e n d ed m a n n er (P e te rs o n a n d Sober, 1962) p rio r to e q u ilib ra tio n in th e d e ­
s ir e d b u ffer.
An io n - e x c h a n g e c e ll u lo s e co lu m n w a s p a c k e d b y f illin g th e g la s s
tu b e to o n e - th ir d i t s v o lu m e w ith s ta r tin g b u ffe r an d th e n a d d in g th e s lu rry
o f e q u ilib r a te d io n - e x c h a n g e m a t e r i a l . The co lu m n s to p c o c k w a s o p e n e d
a n d th e c o lu m n a llo w e d to p a c k w ith o c c a s io n a l ta p p in g to rem o v e o c c lu d e d
a ir b u b b l e s .. E v ery co lu m n w a s e q u ilib r a te d b y a llo w in g b u ffe r to flow
th ro u g h i t o v e rn ig h t a t th e r a te u s e d d u rin g c h ro m a to g ra p h y . C M - c e llu lo s e
vyas r e g e n e r a te d (P e te rs o n a n d Sober, 1962) o n c e ; i . e ., u s e d tw o tim e s ,
th e n d is c a r d e d b e c a u s e i t s io n -e x c h a n g e p ro p e rtie s s e e m e d to c h a n g e w ith
e x te n d e d u s e . A ll e lu tio n s w e re ru n a t room te m p e ra tu re .
' P re p a ra tio n o f G e l F iltr a tio n M ed ia
Two ty p e s o f g e l f iltr a tio n m a te ria l w e re u s e d in t h i s work,; d e x tra n s
o f v a rio u s c r o s s lin k a g e (S ephadex) p u r c h a s e d from P h a rm a c ia F in e C h em i­
c a l s , I n c . a n d p o ly a c ry la m id e (B io -G el P -3 0 0 , 50-150 m esh ) p u rc h a s e d from
B io -R a d -L a b o ra to rie s .
T h e s e m a te r ia ls w e re s u s p e n d e d in th e e lu tin g s o lv e n t to be u s e d an d
w e re a llo w e d to s w e ll a t l e a s t 3 h o u rs in th e c a s e o f g e ls w ith s m a ll p o re
8
s iz e .(S ep h a d e x G -15 a n d G -2 5 ) o r a t l e a s t 18 h o u r s .in th e c a s e o f th e g e ls
w ith la rg e r p o re s i z e . S e p h a d e x p re p a ra tio n s h a d to b e " w a s h e d 1' fre e o f
fin e p a r tic le s to p re v e n t p lu g g in g o f th e ir p a c k e d c o lu m n s . " W a sh in g " w a s
a c c o m p lis h e d in th re e r e p e a te d s te p s : (I) S u s p e n s io n o f S e p h a d e x in th e
e lu tio n s o lv e n t, (2) a 3 0 -m in u te s e ttlin g o f h e a v ie r p a r tic le s , an d (3) d e ­
c a n ta tio n o f th e s u p e r n a ta n t s o lu tio n w h ic h c o n ta in e d fin e p a r t i c l e s . F iv e
o r s ix r e p e titio n s of t h e s e s te p s w e re u s u a ll y s u f f ic ie n t.
A co lu m n w a s p a c k e d b y f illin g th e g l a s s tu b e to o n e - th ir d i t s volum e
w ith e lu tin g s o lv e n t a n d th e n .a d d in g a s lu rry o f th e g e l to b$ u s e d . A fter
th e g e l h a d s e t t l e d to a d e p th o f 1-2 c m , th e e lu tin g s o lv e n t w a s a llo w e d to
flow . The. form ed co lu m n w a s e q u ilib r a te d b e fo re u s e b y p a s s in g s o lv e n t
th ro u g h i t fo r a t l e a s t 12 h o u r s . A ll e lu tio n s w e re p erfo rm ed a t room te m p e rtu re .
E le c tro p h o re s is P ro c e d u re s
A. Io n ic n a tu re of c ru d e m y ro s in a s e
■ The io n ic n a tu re o f th e c o m p o n e n ts in c ru d e m y r o s in a s e w a s c h e c k e d
in a G e lm a n .e le c tr o p h o r e s is a p p a r a tu s . T h is u n it w a s e q u ip p e d w ith S e p ra p h o re III (m icro p o ro u s c e ll u lo s e a c e ta te ) s tr ip s in .s o d iu m b a r b ito l- a c e t a te
b u ffer, pH 8.2, a n d ru n a t 100 v o lts a n d 4 -5 m illia m p s p e r s tr ip fo r tw o h o u rs
a t room te m p e ra tu re . ■A p p ro x im a te ly 2,5 jug o f p ro te in in 0.05 M sodium c i t ­
r a te b u ffer, pH 6.3, w e re a p p lie d p e r s t r i p . P ro te in w a s d e te c te d on th e u n ­
d e v e lo p e d c e ll u lo s e a c e ta te s tr ip w ith Amido S ch w arz s ta i n (100 mg Amido
■S c h w a rz p e r 100 m l 10% a c e t i c a c id ).
9
B . Q u a lita tiv e a n a ly s is fo r h e x o s a m in e
P a p e r e le c tr o p h o r e s is h a s b e e n u s e d to s e p a r a te a n d id e n tif y .h e x o s a m in e s (O hkum a an d S h in o h a ra , 1963). T h is p ro c e d u re w a s u s e d w ith s lig h t
m o d ific a tio n in th e p r e s e n t in v e s t ig a ti o n . A, S p in co M o d el R p a p e r e le c tr o ­
p h o r e s is a p p a r a tu s w a s e q u ip p e d w ith e ig h t B eckm an # 3 2 0 0 4 6 p a p e r s trip s
a n d 0.025 M so d iu m te tr a b o r a te e l e c tr o l y te , pH 9, 2. A fter a llo w in g th is
s y s te m to e q u ilib r a te fo r a n hour, th e sa m p le w a s a p p lie d a s a s trip e w ith a
S p in c o s a m p le s tr ip e r (P art No. 3 0 0 -8 0 5 ), im m e d ia te ly c o v e re d w ith a s trip e
o f 1% (v /v ) a c e t i c a n h y d rid e in a c e to n e s o lu tio n , an d a llo w e d to s ta n d for
10 m in u te s b e fo re s u b je c tin g it to 2 00 v o lts fo r s ix h o u rs a t room te m p e ra ­
tu r e . The p a p e r s tr ip w a s d rie d for fiv e m in u te s a t 9 5 ° C , c o o le d a n d th e N a c e ty l-h e x o s a m in e b a n d s lo c a te d by s ta in in g .w ith p -d im e th y la m in o b e n z a ld e h y d e r e a g e n t (P a rtrid g e , 1948). T h is re a g e n t c a u s e d d a rk e n in g o f th e e n tire
p a p e r s tr ip a f te r 10-15 m in u te s , th e re b y m a sk in g th e b a n d s w h ic h w ere in ­
i t i a l l y e v i d e n t . In th is s y s te m N - a c e ty l- D - g lu c o s a m in e m ig ra te d a b o u t I cm
from th e o rig in to w a rd th e n e g a tiv e .e le c tro d e . N - a c e ty l- D - g a la c to s a m in e
m ig r a te d 'a b o u t 1.5 cm to w a rd th e p o s itiv e e le c tr o d e . O hkum a an d S h in o h ara
(19 63) h a v e re p o rte d th a t N - a c e ty l- D - m a n n o s a m in e m ig ra te d to th e p o s itiv e
e le c tr o d e m ore r a p id ly th a n N - a c e ty l- D - g a la c to s a m in e .
C . H o m o g e n e ity o f p u rifie d m y ro s in a s e
D is k e le c tr o p h o r e s is o n p o ly a c ry la m id e g e ls w a s u s e d to c h e c k hom o­
g e n e ity o f th e p u rifie d m y r o s in a s e . P re p a ra tio n a n d u s e o f 2 - a m in o - 2 - .
(h y d ro x y m e th y l)-1 ,3 p ro p a n e d io l (T R IS )-g ly cin e b u ffer, p o ly a c ry la m id e g e ls
a n d Amido S c h w a rz s ta in in g s o lu tio n fo r p ro te in h a v e b e e n p r e v io u s ly d e ­
s c r i b e d . (D a v is , 1964). A p p ro x im a te ly 1.4 mg o f p u rifie d m y ro s in a s e w ere
10
e q u ilib r a te d o v e rn ig h t w ith T R IS -g ly c in e b u ffer, pH 8.3, b e fo re b e in g s u b ­
j e c t e d to 160 v o lts a t 28 m illia m p s p e r 8 tu b e s fo r 55 m in u te s a t room te m ­
p e ra tu re .
P a p e r C h ro m a to g ra p h y T e c h n iq u e s
F our s o lv e n t s y s te m s w e re u s e d fo r d e s c e n d in g p a p e r c h ro m a to g ra p h y
d u rin g th is in v e s t ig a ti o n . N orm al b u ta n o l- p y r id in e - w a te r (6 :4 :3 , v /v ) w a s
u s e d fo r m o s t ro u tin e s u g a r a n a l y s e s . E th y l a c e ta te - p y r id in e - w a te r (8 :2 :1 ,
v /v ) a n d n - b u ta n o l- a c e tic a c id - w a te r (5 :1 :2 , v /v ) s e rv e d to co n firm th e
id e n tity o f s u g a r s p r e s e n t . U ro n ic a c id c h ro m a to g ra m s w e re ru n in is o p ro p y l
a lc o h o l - p y r id in e - a c e tic a c id - w a te r (8 :8 :1 :4 , v /v ) s o l v e n t . W h atm an #1 f i l ­
te r p a p e r w a s u s e d th ro u g h o u t th is w ork a n d th e e lu tio n s w e re u s u a lly run
fo r 2 4 h o u rs .
R ed u c in g s u g a r s w e re r e v e a le d o n p a p e r c h ro m a to g ra m s b y the- C D -I
r e a g e n t o f G ordon e t a l . (1956). The a ir - d r ie d c h ro m a to g ra m s w e re d ip p e d in
C D - I r e a g e n t, d rie d a t room te m p e ra tu re in th e h o o d an d th e s p o ts d e v e l­
o p e d b y h e a tin g w ith a M a s te r b lo w e r h e a te r. U nknow n s u g a r s w e re c l a s s i ­
fie d by c o lo r o f th e ir C D - I s p o t s . A s u g a r w a s c o n s id e re d id e n tif ie d if ,
w h e n .a know n s u g a r w a s s p o tte d o n th e p a p e r w ith th e u n k n o w n m ix tu r e , in ­
te n s i f ic a tio n o f th e sam e s p o t o c c u rre d in th re e o f th e s o lv e n t s y s te m s m en ­
tio n e d p re v io u s ly .
Amino A cid A n a ly s is
Amino a c id s w e re d e te rm in e d b y th e m e th o d o f M o o re a n d S te in (1954),
w ith a n Auto A n a ly z e r (T ec h n ic o n C h ro m a to g ra p h y C o rp o ra tio n ). S am p les
w e re h y d ro ly z e d in s e a le d e v a c u a te d tu b e s w ith c o n s ta n t b o ilin g
11
h y d ro c h lo ric a c id a t IlO0 C . H y d ro ly z e d s a m p le s w ere f r e e z e - d r ie d , ta k e n
up in d i s t i l l e d w a te r a n d f r e e z e - d r ie d a s e c o n d t i m e . The a q u e o u s s o lu tio n
(0.6 ml) o f th e H C l- fre e h y d ro ly z a te w a s a p p lie d to th e D o w ex 5 0 x 1 2 co lu m n
(60o C) a n d e lu te d from th e co lu m n w ith a pH g ra d ie n t p ro v id e d by 0.05 M
so d iu m c itr a te b u ffe r from pH 2 .8 8 to pH 3.80 a n d by 0.05 M so d iu m c i t r a t e 0.6M so d iu m c h lo rid e b u ffe r from pH 3.80 to pH 5.00.
S e d im e n ta tio n S tu d ie s
The h o m o g e n e ity o f th e m y ro s in a s e p re p a ra tio n w a s s tu d ie d b y re c o rd ­
in g i t s s e d im e n ta tio n a t 59,780 rpiri in a S p in c o M o d el E a n a ly t ic a l u ltr a - .
c e n tr ifu g e e q u ip p e d w ith a s in g le s e c to r c e l l m a in ta in e d a t 3 - 5 ° C . S o lu ­
tio n s w e re p re p a re d by d is s o lv in g th e en zy m e in 0.05 M so d iu m p h o s p h a te
b u ffer, pH 5.7-5.9, a t 5 ° C . The sa m p le w a s p la c e in th e c e n tr ifu g e fo llo w in g
a o n e -h o u r d i a l y s i s a g a in s t th e sam e p h o s p h a te b u ffer. P ic tu r e s o f th e d e ­
v e lo p in g s e d im e n ta tio n p a tte r n w ere ta k e n a t v a rio u s tim e in te r v a ls a f te r th e
ro to r h a d re a c h e d 59,780 rp m .
G a s -L iq u id C h ro m a to g ra p h y o f C a rb o h y d ra te s
G a s - l iq u id c h ro m a to g ra p h y o f th e ir O -tr im e th y ls ily l d e r iv a tiv e s h a s
p ro v id e d s e p a r a tio n a n d id e n tif ic a tio n o f a num ber o f c a r b o h y d ra te s (H ed g ley
a n d O v e re n d , 1960; S w e e l e y e t a l . , 1963; B e n t l e y e t a l , , , 1963; S w e e le y a n d
W alk e r, 1964). T h is te c h n iq u e h a s b e e n r e c e n tly a p p lie d to in v e s tig a tio n s
o n g ly c o p ro te in s (B olton e t a l . , 1965). - A s im ila r a p p ro a c h w a s ta k e n in th e
p r e s e n t in v e s tig a tio n w h e n a n a ly z in g th e m o n o s a c c h a rid e u n its o f p u rifie d
m y r o s in a s e .
12
•A s a m p le (u s u a lly c o n ta in in g 2 0 0 -4 0 0
}Jg o f
c a rb o h y d ra te ) w a s h y d ro ­
ly z e d by re flu x in g w ith 5 ml o f 3.1% (w /w ) m e th a n o lic h y d ro g e n c h lo rid e
p re p a re d by bu b b lin g a n h y d ro u s H C l g a s in to a n h y d ro u s m e th a n o l a t 0 ° C .
U n le s s o th e rw is e s p e c if ie d , re flu x in g l a s t e d fo r 24 h o u r s . A fter e v a p o ra tio n
u n d e r a s tre a m o f n itro g e n g a s , th e r e s id u e w a s d rie d fo r s ix h o u rs in a
v a cu u m d e s ic c a to r e q u ip p e d w ith a n h y d ro u s C aC lg a n d N aO H p e l l e t s . The
r e s u lta n t m e th y l g ly c o s id e s w e re tr e a te d w ith I ml o f m e th a n o l- a c e tic a n h y ­
d rid e (3:1, v /v ) s o lu tio n c o n ta in in g a few m illig ra m s o f s ilv e r a c e t a t e . T h is
tr e a tm e n t l a s t e d 24 h o u rs a t room te m p e ra tu re a n d w a s p e rfo rm ed in o rd e r;to
a c e ty la te a n y m a te ria l th a t m ig h t h a v e b e e n d e - N - a c e ty la te d d u rin g h y d ro ­
l y s i s , (W hite, 1940). F o llo w in g f iltr a tio n th ro u g h s in te r e d g la s s , th e f iltr a te
w a s e v a p o r a te d in a s tre a m o f n itro g e n g a s a n d d rie d in a v a cu u m d e s ic c a to r
fo r 24 h o u r s . T rih ie th y ls ily l e th e r s o f th e g ly c o s id e m ix tu re w e re p re p a re d
b y a -1 0 -m in u te tr e a tm e n t w ith 0.10 ml o f a p y r id in e - tr im e th y lc h lo r o s ila n e h e x a m e th y ld is ila z a n e (5 :1 :1 , v /v ) m ix tu r e . (R eag en t g ra d e p y rid in e w a s r e ­
d i s t i l l e d from BaO a n d s to re d o v e r KOH p e lle ts p rio r to u s e . T rim e th y lr ...
c h lo r o s ila n e a n d h e x a m e th y ld is ila z a n e w e re u s e d a s r e c e iv e d from K an d K
L a b o ra to rie s , In c .). The tr i m e th y ls ily la tio n m ix tu re c o n ta in e d a g ra y to
brow n p r e c ip ita te w h ic h w a s p ro b a b ly am m onium c h lo rid e a n d re d u c e d s ilv e r
io n . K agan a n d M ab ry (1965) h a v e re p o rte d th a t th is p r e c ip ita te d o e s n o t
in te rfe re w ith q u a n tita tiv e r e s u l t s o b t a i n e d .
A ll a n a ly s e s w e re p e rfo rm ed in a F a n d M B io m e d ica l M o d el 400 g a s
c h ro m a to g ra p h e q u ip p e d w ith te m p e ra tu re p ro g ram m in g , a fla m e io n iz a tio n
d e te c to r a n d a U - s h a p e d g l a s s co lu m n (1 0 5 x 0 .4 cm) p a c k e d w ith 3.8% S E -30
13
s ilic o n e o h G a s C hrom e Q (1 0 0 -1 2 0 m esh ). H eliu m s e rv e d a s c a r r ie r g a s
a n d w a s m a in ta in e d a t a flow r a te o f 7 0 -7 2 ml p e r m in u te . A liq u o ts (0 .8 -1.1
jjl) o f th e tr im e th y ls ily la tio n m ix tu re w e re in je c te d in to th e co lu m n a t IOO0 C
a n d , a f te r a 2 -m in u te d e la y , th e co lu m n te m p e ra tu re w a s program m ed (3°C
p e r m in u te) to 2 OO0 C . Q u a lita tiv e a n a ly s e s w e re in itia te d a t 150°C a n d , a f ­
t e r a 1 0 -m in u te d e la y , program m ed (7.5 °C p e r m in u te) to 2 QO0 C . Q u a n tita ­
tiv e d e te r m in a tio n s re q u ire d g re a te r s e p a r a tio n o f p e a k s p ro v id e d by th e
lo w e r i n itia l te m p e r a tu r e .
The p e a k s o b ta in e d w e re id e n tif ie d b y c o m p arin g th e ir re te n tio n tim e s
w ith th o s e o f k now n s u b s ta n c e s p re p a re d in th e m an n er d e s c r ib e d a b o v e .
R e la tiv e a m o u n ts o f th e s u g a r s p r e s e n t in v a rio u s u n k now n p re p a ra tio n s
w e re d e te rm in e d d ir e c tly from in te g r a to r r e a d in g s o n a S a rg e n t M odeT SR
re c o rd er..
EXPERIMENTAL
E x tra c tio n an d P u r ific a tio n o f M y ro s in a s e
T h irty -o n e k ilo g ra m s o f y e llo w O rie n ta l m u s ta rd s e e d , B ra s s ic a
J u n c e a , w e re g ro u n d in to a c o a r s e m e a l an d d e f a tte d w ith S k e lly B s o lv e n t
in. a. S o x h le t. e x tra c to r. A fter a te n - h o u r a ir d ry in g , th e 10 k g o f d e fa tte d
m e a l w e re m ille d to a pow der, s u s p e n d e d in 91 lite r s o f d i s t i l l e d w a te r
(440 C) a n d m e c h a n ic a lly s tirre d for s e v e n h o u rs . A m ajo r p o rtio n o f th e
d e b ris in th e r e s u ltin g s lu rry w a s re m o v e d by c e n tr if u g a tio n . The re m a in in g
d e b ris w a s a llo w e d to s e t t l e fo r 12 h o u rs a t 5 ° C . A c le a r a m b e r-c o lo re d s u -.
p e rn a ta n t s o lu tio n r e s u lte d a n d w a s c o lle c te d b y d e c a n ta tio n . T h is s u p e r­
n a ta n t s o lu tio n p lu s o n e w a te r w a s h c o n s titu te d th e e n z y m e -c o n ta in in g
w a te r e x t r a c t . The w a te r e x tr a c t (44 lite r s ) w a s c o o le d .to 5 ° 0 a n d 'tr e a te d
w ith 10.7 kg (N H ^gSO ^ to g iv e 40% s a tu r a tio n . P r e c ip ita te d p ro te in w a s r e ­
m o v e d .in a W e s tp h a lia c e n tr ifu g e a n d d is c a r d e d . Ammonium s u lf a te (11.6 kg)
w a s a d d e d to th e 40% s a tu r a te d s o lu tio n to g iv e 80% s a t u r a t i o n . The
e n z y m e -c o n ta in in g p r e c ip ita te w a s c o lle c te d by c e n trifu g a tio n ,, d is s o lv e d in
22 li t e r s o f d i s t i l l e d w a te r an d d ia ly z e d fo r 24 h o u rs a g a in s t c o ld running,
ta p w a t e r .. An e q u a l v o lu m e o f a b s o lu te e th a n o l (S0 C) w a s s lo w ly a d d e d to
th e p ro te in s o lu tio n (15°C). A fter m ix in g fo r 30 m in u te s, th e p r e c ip ita te w a s
re c o v e re d by c e n tr if u g a tio n , d is s o lv e d in d i s t i l l e d w a te r (5 lite r s ) an d d rie d
u n d e r fo rc e d a ir a t room te m p e ra tu re in alum inum t r a y s . A fter 36 h o u rs, 87
g ra m s o f c le a r , a m b e r - c o lo r e d , fla k y c ru d e m y ro s in a s e w e re o b ta in e d .
F r a c tio n a tio n o f c ru d e m y ro s in a s e o n D E A E -ce llu lo se w a s u s e d in a n
a tte m p t to s e p a r a te th io g lu c o s id a s e a n d s u lf a ta s e , a c ti v ity a s p re v io u s ly
15
re p o rte d (G a in e s a n d .G o e rin g , 1960). P ro te in w a s re c o v e re d a s a s in g le
f r a c tio n w h en e lu te d from th e co lu m n w ith 0.05 M so d iu m c i t r a t e bu ffer,
pH 6.3. E le c tr o p h o r e s is o n c e ll u lo s e a c e t a t e s tr ip s .(See " M a te r ia ls and
M e th o d s " ) sh o w e d t h i s p ro te in in pH 6.3 s o lu tio n to b e h a v e a s a c a tio n .
T h is in fo rm a tio n s u g g e s te d th e u s e o f io n - e x c h a n g e te c h n iq u e s o n C M c e llu lO s e .
F o u r-g ra m p o rtio n s o f c ru d e m y ro s in a s e w e re d is s o lv e d in a p p ro x i­
m a te ly 75 m l o f 0.05 M so d iu m c itr a te b u ffer, pH 4.3, a n d p la c e d o n C M c e H u lo s e c o lu m n s (3 0 x 3 .4 cm) e q u ilib r a te d w ith th e sam e b u ffer. P ro te in
w a s e lu te d from th e c o lu m n s (2 m l/m in ) in th r e e s te p s : (I) 0.05 M .sodium
c i tr a te b u ffer, pH 4.3, (2) 0.05 M so d iu m c itr a te bu ffer, pH 5.8, a n d (3)
0.05 M. so d iu m c i t r a t e - 1 M so d iu m c h lo rid e b u ffer, pH 6.8. F ig u re I i l l u s ­
t r a t e s th e ty p ic a l e lu tio n p a tte r n o b ta in e d . M a jo r a m o u n ts o f en zy m e a p ­
p e a r e d in th e p ro te in e lu te d a t pH 5.8 a n d t h i s w as d ia ly z e d , c o n c e n tra te d
b y b lo w in g a ir o v e r th e d ia ly s is b a g c o n ta in in g th e en zy m e, a n d freezerd rie d . A p p ro x im a te ly 2 g o f e n z y m e -c o n ta in in g p ro te in (C M C -2 ) r e s u lte d
from f r a c tio n a tio n o f 30 g o f c ru d e m y r o s in a s e . S e d im e n ta tio n in a n u ltr a ­
c e n tr ifu g e r e v e a le d a t l e a s t th r e e c o m p o n e n ts in C M C -2 (F ig u re 2A).. G el
f iltr a tio n through- S e p h a d e x G -2 0 0 (6 0 x 3 .9 cm) e q u ilib ra te d w ith 0.05 M s o ­
dium c i tr a te b u ffer, pH 6.1, s e p a r a te d C M C -2 in to a t l e a s t th r e e fr a c tio n s
,(F igure 3). T e n - m illilite r a liq u o ts w e re c o lle c te d a t a r a te o f 30 ml p e r h o u r.
Enzym e a c ti v ity a p p e a re d e n tir e ly w ith th e p ro te in e lu te d in th e f ir s t p e a k .
An iv o r y - ta n , fla k y m a te ria l (305 mg) w a s o b ta in e d a f te r d ia ly z in g , c o n c e n ­
tr a tin g a n d f r e e z e - d r y in g th is .p o rtio n . H o m o g e n e ity o f th e e n zy m e w a s a g a in
c h e c k e d in a n u ltr a c e n tr if u g e (F igure 2J3). I o n - e x c h a n g e c h ro m a to g ra p h y w a s
16
0.05 M
C itr a te
pH 4.3
0.05 M
C itr a te
pH 5.8
0.05 M C itra te
— I M N aC l —
pH 6.8
C M C -2
•
0.6
F ra c tio n N um ber
F ig u re I . F r a c tio n a tio n o f c ru d e m y ro s in a s e by s te p - w is e e lu tio n from
C M -c e llu lo s e .
17
a tte m p te d o n D E A E -c e llu lo se u s in g so d iu m c itr a te b u ffer, pH 6.1, a s th e
e lu tin g a g e n t; b u t a g a in , th is f a ile d to s e p a r a te th io g lu c o s id a s e from s u lf a ta s e a c ti v ity (F igure 4).
F ig u re 2 . S e d im e n ta tio n p a tte r n s o f m y ro s in a s e p r e p a r a tio n s . C e n trifu g e
s p e e d w a s 59,780 rp m . Tim e o f p h o to g ra p h s a f te r a tta in in g s p e e d g iv e n in
p a r e n th e s e s : I (15 m in), 2 (23 m in), 3 (31 m in), 4 (3 m in), 5 (11 min),
6 (19 m in). A. E x p e rim en t c o n d u c te d a t 5°C in 0.1 M p h o s p h a te buffer,
pH 5.7, w ith C M C -2 c o n c e n tr a tio n o f 9 m g /m l. B . E x p e rim en t c o n d u c te d a t
S0 C in 0.05 M p h o s p h a te b u ffer, pH 5.9, w ith p u rifie d m y ro s in a s e c o n c e n tr a ­
tio n o f 6 m g /m l.
18
F ra c tio n N um ber
F ig u re 3 .
G e l f iltr a tio n o f C M C -2 o n S e p h a d e x G - 2 0 0 .
19
F ra c tio n N um ber
F ig u re 4 . E lu tio n o f p u rifie d m y ro s in a s e from D E A E -ce llu lo se w ith 0.05 M
so d iu m c itr a te buffer, pH 6.1.
D isk e le c tr o p h o r e s is p erfo rm ed o n p o ly a c ry la m id e g e l in 0.10 M TRISg ly c in e b u ffer, pH 8.3, re v e a le d h e te r o g e n e ity in th e e n zy m e p re p a ra tio n
(F igure 5). E ach o f th e fiv e b a n d s a p p e a re d to e x h ib it m y ro s in a s e a c tiv ity .
E nzym e a c ti v ity w a s a s c e r ta in e d by c u ttin g th e b a n d s from a g e l u s in g a
s ta in e d g e l a s r e f e r e n c e . E ach b an d w a s s u s p e n d e d in 1.0 ml 0.05 M so d iu m
c i tr a te b u ffer, pH 6.1, w h ic h c o n ta in e d 10 mg o f s in ig rin a n d in c u b a te d a t
3 7 °C for 36 h o u r s . E nzym e a c ti v ity w a s d e te c te d by s m e llin g th e in c u b a tio n
v ia l.
20
F ig u re 5 . D is k e le c tr o p h o r e s is o f p u rifie d m y ro s in a s e o n p o ly a c ry la m id e
g e l a t pH 8.3.
No fu rth e r p u rif ic a tio n o f th e en zy m e w a s a tte m p te d . T a b le I sum m a­
r iz e s th e p u r if ic a tio n o f m y ro s in a s e from a s e c o n d b a tc h o f m u sta rd s e e d
(36 kg) tr e a te d in th e a b o v e m anner. The en zy m e p re p a ra tio n from g e l f i l t r a ­
tio n on S e p h a d e x G -2 0 0 w a s re fe rre d to a s " p u rifie d m y ro s in a s e " d u rin g th is
w o rk .
C h e m ic a l C o m p o sitio n of M y ro s in a s e
A. M o is tu re c o n te n t
D u p lic a te s a m p le s o f p u rifie d m y ro s in a s e w ere a n a ly z e d for m o istu re
c o n te n t by a n in d ir e c t w e ig h t- lo s s m e th o d . The s a m p le s w e re w e ig h e d in a
p re v io u s ly w e ig h e d v ia l a n d p la c e d in a n o v e n a t 104°C for 12 h o u r s . F o l­
lo w in g a o n e -h o u r c o o lin g p e rio d in a d e s ic c a to r th e s a m p le s w e re w e ig h e d
a n d d rie d a n a d d itio n a l s ix h o u r s . No a d d itio n a l w a te r w a s l o s t d u rin g th e
s e c o n d d ry in g tim e . D u p lic a te s a m p le s d rie d for 17 h o u rs a t IOO0 C in a
v acu u m o v e n g a v e c o m p a ra b le r e s u l t s . M o is tu re c o n te n t o f th e en zy m e p re ­
p a ra tio n is g iv e n in T ab le II .
Table I . Purification o f B rasslca iuncea M yrosinase
Volume
Step
(liters)
U nitsa/m l
Tb
Sc
Total U nits
T
3.87 x IO"1 6.12 xlO "1 57,700 91,200
I . W ater Extract
149
Z . 40-80% (NH4)2S04
32.0
1.65
3 . 50% Ethanol
Precipitation
4.40
5.42
4 . C M -C ellulose
2.84e
2.77
1.95
7,900
5 . G el Filtration
Sephadex G-200
0.92®
5.00
3.62
4,600
Precipitation
S
•
Proteincl
(mg/ml)
Specific Activity
(units/m g-protein)
T
S
Specific Activity
Ratio
T/S
Yield
(%).
T
S
Purification
T '
S
30.60.
U S x lO * 2
2.00 x IO"2
0.64
100
100
I
I
2.42
52,900 77,400
26.40
62 6 x IO"2
9.18 x IO"2
0.68
91.6
85.0
42
4.6
7.15
23,800 31,400
15.00
3.61 xlO "1
4.76 x 10"1
0.76
41,2
34.5
28.0
24.0
5,550
1.56
1.78
1.25
1.42
13.7
6.1
139
62
3,330
1.57
320
2.30
1.40
8.0
3.6
250
HS
a Unit defined a s th a t amount of enzyme required to hydrolyze 5 mg sinigrin per.hour a t 3 7 °C .
b T hioglucosidaSe activity. G lucose deter­
mined by quantitative paper chromatography in Step I . .D initrosalicylic acid reag en t w as used to determine glucose in a ll other s te p s .
c S ulfatase activity. Sulfate determ ined turbidim etrlcally a s BaSO^.
^ Protein determ ined by method o f Lowry e t a l . (19S I).
extrapolated from portions o f the 50% ethanol precip itate which were further p u rified .
e Value
22
T a b le I I .
C h e m ic a l C o m p o sitio n o f M y ro s in a s e
P e rc e n t
N um ber o f
M e th o d s U se d
P ro te in
55.8
2
C a rb o h y d ra te
13.3
2
W a te r
7.4
2
C itr a te (P o s s ib le )3
8.2
I
Sodium io n ( P o s s ib le )'3
6.4
I
T o ta lc
91.1
a E s tim a tio n b a s e d on a m o u n ts o f a rg in in e a n d ly s in e in m y r o s in a s e .
b E s tim a tio n b a s e d on a m o u n ts o f a s p a r tic , g lu ta m ic , s i a l i c a n d c itr ic a c id
p r e s e n t . c A n a ly s e s fo r l i p i d , P, in o s ito l w e re a l l n e g a tiv e .
B . P ro te in -a m in o a c id c o n te n t
T o ta l n itro g e n (8.92%) in p u rifie d m y ro s in a s e w a s d e te rm in e d b y th e
m ic ro -K je ld a h l m eth o d a t G a lb ra ith L a b o ra to rie s , I n c ., K n o x v ille , T e n n e s s e e .
T he u s u a l c o n v e r s io n f a c tp r (6.2 5) w a s u s e d in c a lc u la tin g a p e rc e n t p ro te in
from th e to t a l n itro g e n c o n te n t o f th e e n zy m e p r e p a r a tio n . T h is r e s u lt is in ­
c lu d e d in T a b le II .
A mino a c id s w e re a n a ly z e d b y th e m e th o d d e s c r ib e d in " M a te ria ls a n d
M e th o d s " . P e rc e n t p ro te in (55.5%) b a s e d o n am ino a c id s re c o v e re d d u rin g
th i s a n a ly s is s u b s ta n tia te d th e r e s u lts o b ta in e d by th e m ic ro -K je ld a h l
m e th o d . T h ree m y ro s in a s e s a m p le s w e re h y d ro ly z e d fo r 24 h o u rs an d o n e
sa m p le for 72 h o u rs p rio r to a n a l y s i s . T he 72-h o u r h y d r o ly s is r e v e a le d p a r­
t i a l d e s tr u c tio n o f som e am in o a c i d s . C o rre la tio n o f r e s u l t s from th e
23
7 2 -h o u r and. 2 4 - hour, h y d ro ly z a te s a llo w e d e x tra p o la tio n to z ero tim e o f h y ­
d r o ly s is w h en d e te rm in in g a m o u n ts o f t h e s e la b ile am in o a c i d s . T ry p to p h an
w a s c o m p le te ly d e s tr o y e d d u rin g h y d r o ly s is a n d , th e re fo re , w a s d e te rm in e d
in d e p e n d e n tly by th e s p e c tro p h o to m e tric m eth o d o f G o odw in a n d M orton
(1946). The am ino a c id c o m p o s itio n o f m y ro s in a s e a s d e te rm in e d in th is in ­
v e s ti g a tio n is g iv e n in' T ab le II I.
C . C a rb o h y d ra te c o n te n t
A n a ly s e s o f c a rb o h y d ra te in th e p r e s e n c e o f p ro te in h a v e u s u a lly b e e n
a c c o m p a n ie d by m any p ro b le m s a r is in g from c h e m ic a l in te r a c tio n b e tw e e n •
am ino a c id s .a n d c a rb o h y d ra te d u rin g h y d ro ly s is ( G o tts c h a lk , 1963). In a d d i­
t io n , s e p a r a tio n o f th e r e s u lta n t m o n o s a c c h a r id e s from t h e s e s id e p ro d u c ts
a n d am ino a c id s h a s b e e n tim e -c o n s u m in g a n d p ro n e to e x p e r im e n ta l e rro r.
In th e p r e s e n t in v e s tig a tio n a m e th o d (s e e " M a te ria ls a n d M e th o d s " ) w a s
u s e d w h ic h o v e rc a m e som e o f th e s e p ro b le m s . H y d ro ly s is o f m y ro s in a s e
>
w ith m e th a n o lic H C l g a v e th e fre e m o n o s a c c h a r id e s a s th e Ir m e th y l g ly c o ­
s id e s a n d p a r tia lly d e g ra d e d p r o te in . S u b s e q u e n t fo rm a tio n o f th e ir O tr im e th y ls ily l e th e r s a n d a n a ly s is b y g a s - l i q u i d c h ro m a to g ra p h y a llo w e d
s e p a r a tio n , id e n tif ic a tio n a n d d e te rm in a tio n o f th e q u a n tita tiv e r a tio s o f
s u g a r s p r e s e n t in th e en zy m e p r e p a r a tio n .
S a m p le s o f m y ro s in a s e w e re h y d ro ly z e d fo r p e rio d s o f 24, 48 an d 72
h o u rs b e fo re p re p a rin g th em for g a s - l i q u i d c h ro m a to g ra p h y . T y p ic a l ch ro m a ­
to g ra m s o b ta in e d from t h e s e p re p a ra tio n s a re sh o w n in F ig u re s 6, 7 a n d 8.
T a b le IV g iv e s v a lu e s , a s d e te rm in e d b y .g a s - liq u id c h ro m a to g ra p h y , for th e
r e la tiv e p e r c e n ta g e s o f e a c h s u g a r u n it p r e s e n t in th e to ta l c a r b o h y d r a te .
T a b le I I I .
Amino A cid
A s p a rtic a c id
T h re o n in e .
S e rin e
G lu ta m ic a c id
P ro lin e
G ly c in e
A la n in e
V alin e
H a I f - c y s tin e .
M e th io n in e
I s o le u c in e
L e u c in e
T y ro sin e
P h e n y la la n ih e
Ammonia
L y sin e
H is tid in e
A rg in in e
T ry p to p h a n 6
T o ta ls
Amino A cid C o m p o sitio n o f M y ro s in a s e a
Amino A cid
Amino A cyl
R e sid u e
(g/lOOg G ly c o p ro te in )
.24 hr. 72 hr. O h r .^ (g/lOOg. G ly c o p ro te in )
10.16
3.49
5.09
15.77
6.68
5.15
4.06
4.44
1.00
1.73
3.08
5.91
3.71
4.2 0
1.79d
3.64
2.04
5.25
—
10.17
3.94
4.58
16.99
5.76
4.98
3.64
5.32
85.40
85.41
—
—
3.96
6.90
2.85
4.38
1.48d
4.71
2.08
5.15
—
10.1.6
3.94
5.35
16.99
7.14
5.24
4.26
5.32
1.00
1.73
3.96
6.90
4.13
4.38
1.9 4d
4.71
2.08
5.35
1.83
94.47
8.79
3.34
4.44
14.91
5.41
3.99
3.40
4.51
0.86
1.53
3.42
5.95
3.72
. 3.90
—
4.13
1.83
4.80
1.67
80.60
.
Amino A cid
N itro g e n
(m ol/10,0OOg G ly c o p ro te in )
T o tal N) c
(% o f
7.6
3.3
5.1
11.5
6.2
7.0
4.8
4.5
0.8
1.2
3.0
5.3
.2.3
2 .6
—
3.2
1.3
3.1
0.9
— —
8 .2 5 .
'
3.55
5.47
12.56
6.71
7.56
5.17
4.93
0.92
1.23
3.24
5.70
2.45
2.85
------ --
6.90
4.32
13.26
1.93
97.00
a. C o rre c te d to in c lu d e o n ly p r o te in -c a rb o h y d ra te c o n te n t o f p re p a ra tio n . ^ R e s u lts e x p r e s s e d a s
e x tr a p o la tio n s to z ero tim e h y d r o ly s is o r a t m ax im al re c o v e ry . c 12.98% N in p r e p a r a tio n . d T his
v a lu e o m itte d from t o t a l . e : D e te rm in e d b y th e m eth o d o f G o odw in an d M o rto n (1946).
6
1-----------------------------------—
i
5
IO
—
h
15
7
--------------------------—
20
i
25
-------------1
30
r■
■
-------- f ■
35
M in u te s
F ig u re 6. G a s ch ro m ato g ram of th e tr im e th y ls ily l d e r iv a tiv e s from th e 2 4 -h o u r m y ro s in a s e h y d ro ly z a t e . (I) ? M e th y l c itr a te (2) M e th y l ,6- L - a r a b in o s id e
(3) M e th y l ,5 -L -fu c o s id e (4) M e th y l
tf - D - x y lo s id e (5) M e th y l /S -D -x y lo s id e (6) M e th y l O L-D -m annoside (7) M e th y l O -D -g a la c to s id e
(8) M e th y l /3 - D - g a la c to s id e (9) M e th y l (X -D -g lu c o s id e (10) M e th y l/3 - D - g lu c o s id e (11) M eth y l
2 - a c e ta m id o - 2 - d e o x y - D - g lu c o s id e (12) N -a c e ty l- n e u ra m in ic a c id .
M in u te s
F ig u re 7. G a s ch ro m a to g ra m o f th e tr i m e t h y l s i l y l d e r i v a t i v e s from th e 4 8 -h o u r m y r o s in a s e h y d ro z a t e . S ee Fig u re 6 for i d e n t i f i c a t i o n o f p e a k s .
M in u te s
F ig u re 8 . G a s ch ro m ato g ram o f th e tr i m e t h y l s i l y l d e r i v a t i v e s from th e 7 2 -h o u r m y ro s in a s e h y d ro z a t e . S ee Fig u re 6 for i d e n t i f i c a t i o n of p e a k s .
28
----MMMLJl-. --
T a b le IV.
u
rn I
——
ir^P
i ■
■
I
C o m p o s itio n o f th e C a r b o h y d ra te A s s o c i a t e d w ith M y r o s i n a s e .
H y d ro ly z e d S u g ar a
(g/lOOg C a rb o h y d ra te )
2 4 hr. 48 hr, 72 hr.
S u g ar
L -arab in o se
L -fucose
D -x y lo se
D -m anngse
D -g a la cto se
D -g lu c o se
D -g lu co sam in e ^
S ialic a c id e
T o ta ls
<
W».
A verage Sugar
C o n te n t
(g/lOOg C a rb o h y d ra te )
15.2
2.6
5.9
(26.8)5
(31.5)c
10.9
17.6
2.4
4.8
31.9
28.2
13.9
16.2
2.2
(10.3)0
33.0
25.9
11,7
16.3
.2.4 .
5.5
32.5 '
27.0 .
12.2
.2.7
0.9
1.6
4.9
■ 1.1
—
—
4.9
' 100.5
99.9
100.2
. 102.4
A lte rn a te
Q u a lita tiv e
A n a ly s is b
+
+
(+)
+'
+
+
■T—
a O b ta in e d by g a s - l i q u i d c h ro m a to g ra p h y of r e s i d u e s from m y r o s in a s e as.
d e s c r i b e d in t e x t , b O b ta in e d b y p a p e r c h ro m a to g ra p h y o f C M C -2 h y d ro ly z a t e a s d e s c r i b e d in text;: + = i d e n t i f i e d , (+) = t e n t a t i v e id e n tity ,
= ho t ■
d e t e c t e d . c V a lu e s in p a r e n t h e s e s no t u s e d in c a l c u l a t i n g a v e r a g e s u g a r
■ c o n t e n t . ^ A n a ly z e d a s t h e N - a c e t y l d e r i v a t i v e . e • N - e c e ty l- n e u r a m in i c
a c id .
C M - c e l l u l o s e a n d S e p h a d e x G-200. m ig h t h a v e , b e e n d e g r a d e d in som e
m a n n e r w h ile b e in g u s e d in th e p u r if ic a tio n of m y r o s in a s e a n d , th e re fo re ,
m ig h t h a v e c o n ta m in a te d t h e e n zy m e p r e p a r a t io n w ith c a r b o h y d r a t e . In o rd e r
to c h e c k t h i s p o s s i b i l i t y , a b la n k w a s p r e p a r e d by s u b j e c t i n g a s am p le
w h ic h d id ho t c o n t a i n p r o te in to th e p r o c e d u r e s w h ic h w e re u s e d to p u rify
c ru d e m y r o s i n a s e . A n a ly s is of t h i s b la n k b y g a s - l i q u i d c h ro m a to g ra p h y
s h o w e d o n ly m inor a m o u n ts o f D - g l u c o s e to be p r e s e n t .
A s t a n d a r d i z a t i o n c u rv e for t h e p h e n o l - s u l f u r i c a c i d m e th o d for t o t a l
c a r b o h y d r a te a n a l y s i s w a s p r e p a r e d b y u s in g a c o m p o s ite s t a n d a r d c o m p o s e d
29
o f t h e s u g a r s (m inus s i a l i c a c id ) in t h e i r w e ig h t r a t i o s a f t e r 2 4 - h o u r h y ­
d r o l y s i s a s g iv e n in T a b le IV. P e r c e n t c a r b o h y d r a te in m y r o s in a s e w a s d e ­
te rm in e d in t r i p l i c a t e a n d is r e c o r d e d in T a b le I I . E s tim a te s (a p p ro x im a te ly
1 1.5%) from gas. c h ro m a to g ra m s s u b s t a n t i a t e d t h e s e r e s u l t s .
D a ta from g a s - l i q u i d c h ro m a to g ra p h y s u p p o rte d e a r l i e r q u a lita tiv e
e v i d e n c e g a in e d by a c i d h y d r o l y s i s o f C M C - 2 , s e p a r a t i o n b y m e a n s o f io n e x c h a n g e r e s i n s ,(Simkin e t Eil., 1964), a n d s u b s e q u e n t , a n a l y s i s of the. c a r b o ­
h y d r a te r e s i d u e s b y p a p e r c h ro m a to g ra p h y a n d p a p e r e l e c t r o p h o r e s i s . O n e
h u n d re d m illig ra m s o f C M C -2 w e re d i s s o l v e d in 15 ml 0.2 N H C l c o n ta in in g
0.9Sg' D o w ex 5 OWx 8 (H+) io n - e x c h a n g g r e s i n a n d r e f lu x e d for 24 h o u r s . The
m ix tu re w a s q u a n t i t a t i v e l y f i l t e r e d th ro u g h a s i n t e r e d g l a s s fu n n e l unto a
D o w ex 5 OW x 12 (H+) c o lu m n (18x1.6 cm) w h ic h d rip p e d u n to a D o w ex 2 x 8
(HCOO- ) co lu m n ( 2 4 x 1.6 cm). After fiv e 5 -m l w a s h e s th e fu n n e l w a s r e ­
m o v e d a n d th e tw o - c o lu m n s y s te m e lu te d w ith d i s t i l l e d w a te r. The f ir s t 30
ml w e re d i s c a r d e d a n d th e n e x t 120 ml c o l l e c t e d . T h is n e u tr a l s u g a r fr a c tio n
w a s e v a p o r a te d ip a r o ta r y e v a p o ra to r, t r a n s f e r r e d in 6-7. ml of a q u e o u s s o ­
lu t i o n to a s m a ll v i a l a n d d rie d u n d e r a s tr e a m of n itr o g e n g a s . The two
i o n - e x c h a n g e c o lu m n s w e re d i s c o n n e c t e d a n d m o u n te d for in d iv id u a l e l u ­
t i o n . H e x o s a m in e a n d som e am in o acid s, w e re e lu te d from th e D ow ex .
5 OWx 12 w ith .2 N H C l . The f i r s t 20 ml o f e l u a n t w e re d i s c a r d e d a n d th e
n e x t 120 ml w e re r e t a i n e d a n d c o n c e n t r a t e d in th e sam e m a n n e r a s th e n e Ul­
tra I f r a c t i o n . T h is w a s th e h e x o s a m f n e f r a c t i o n . The D o w ex 2 x 8 colum n
w a s e lu te d w ith 1.2N form ic a c i d to re m o v e a n y u ro n ic or s i a l i c a c i d s t h a t
m ay h a v e b e e n r e t a i n e d from th e o r ig i n a l h y ^ r o l y z a t e . A fter d i s c a r d i n g th e
f i r s t 2 0 ml of e l u a n t , t h e n e x t 100 ml c o n s t i t u t e d th e u ro n ic a c i d fr a c tio n
30
a n d w e re c o n c e n t r a t e d in th e u s u a l manner.' M e th o d s w h ic h h a v e b e e n p r e ­
v i o u s l y d e s c r i b e d w e re u s e d to a n a l y z e e a c h o f t h e s e th r e e f r a c t i o n s . N e u ­
t r a l s u g a r a n d u ro n ic a c i d f r a c tio n s w e re a n a l y z e d by p a p e r c h ro m a to g ra p h y
a n d th e h e x o s a m in e f r a c tio n w a s s u b j e c t e d to p a p e r e l e c t r o p h o r e s i s . All
m ig r a tin g s p o ts w e re i d e n t i f i e d a n d th e r e s u l t s a re i n c lu d e d in Table IV.
- D. M i s c e l l a n e o u s a n a l y s e s
P r o te i n , c a r b o h y d r a te a n d m o is tu re a c c o u n t e d for a b o u t 76% of th e
t o t a l w e ig h t o f f r e e z e - d r i e d p u rif ie d m y r o s i n a s e . To a c c o u n t for th e re m a in ­
in g 24% som e a d d i t i o n a l a n a l y s e s w e re a t t e m p t e d .
L ip id m a te r ia l f r e q u e n tly a c c o m p a n ie s p ro te in d u rin g p r o te in is o la tio n !
S in c e th e m u s ta r d s e e d h a d a h ig h o il c o n te n t (a p p ro x im a te ly 30%), s u c h
m a te r ia l c o u ld l i k e l y b e p r e s e n t in th e e n z y m e p r e p a r a t i o n . A s a m p le of
p u r if ie d m y r o s in a s e (1.87 mg) w a s h y d r o ly z e d w ith m e th a n o lic H C l in th e
m a n n e r u s e d d u rin g p r e p a r a t io n for g a s - l i q u i d c h ro m a to g ra p h y of c a r b o h y ­
d r a t e s . A fter h y d r o l y s i s t h e d rie d r e s i d u e w a s d i s s o l v e d in 0.05 ml
c h lo ro fo r m -m e th a n o l s o l v e n t (2 :1 , v / v ) . A 3 -jjl s am p le a n d a l->ll s a m p le o f
t h i s s o lu tio n w e re s p o t t e d o n a t h i n - l a y e r c h ro m a to g ra p h y p l a t e c o a te d w ith
A b s o r b o s i l - 3 c o n ta i n in g 0.02% R hodam ine 6 - G . S a m p le s o f s t e a r i c a c id a n d
v a r io u s s ta n d a r d f a t t y a c i d e s t e r s w e re s p o t t e d o n th e s a m e p l a t e . The
p l a t e w a s d e v e lo p e d for 30 m in u te s in a h e x a n e - d i e t h y l e t h e r (7:3, v /v ) s o l ­
v e n t . D e t e c t i o n of m ig ra tin g s p e c i e s w a s m ad e u n d e r u l t r a v i o l e t lig h t a n d
a l s o b y e x p o s in g th e p l a t e s to io d in e v a p o r. The s o lv e n t c a u s e d e x t e n s i v e
m ig r a tio n o f t h e f a t t y a c i d e s t e r s a n d a s l i g h t m ig ra tio n o f th e s t e a r i c a c i d .
No m ig ra tin g c o m p o n e n ts e m e rg e d from th e m y r o s in a s e h y d ro ly z a te .. To c o n ­
firm t h e s e r e s u l t s a m o d if ic a tio n o f th e li p i d e x t r a c ti o n p ro c e d u re of F o lch
31
e t a l . (1957) w a s t h e n a t t e m p t e d . A s u s p e n s i o n o f m y r p s in a s e (6.75 mg) in
4.5 ml c h lo r o f o r m - m e th a n o l s o l v e n t (2 :1 , v / v ) w a s a g i t a t e d a t in t e r v a l s for 2
h o u rs a t 5 ° C . A fter c e n t r i f u g a t i o n a t s lo w s p e e d , th e s u p e r n a t a n t s o lu tio n
w a s d e c a n t e d in to a w e ig h e d v i a l a n d d rie d in a s tre a m of n itr o g e n g a s . A
f r e s h p o rtio n o f s o l v e n t w a s a d d e d to th e p r e c i p i t a t e a n d th e p ro c e d u re r e ­
p e a t e d 3 t i m e s . E a c h of th e 3 s u p e r n a t a n t s o l u t i o n s w a s c o m b in e d w ith th e
r e s i d u e o f p r e v io u s s u p e r n a t a n t s o l u t i o n s . A fter dry in g 8 h o u rs in a v a cu u m
d e s i c c a t o r , no r e s i d u e w a s d e t e c t e d b y w e i g h i n g . T h erefo re, lip id w a s r u le d
o p t a s a m a jo r c o m p o n e n t in th e m y r o s in a s e p r e p a r a t io n .
I n o s i t o l is a l s o a com m on c o m p o n e n t o f p la n t s e e d s a n d i s u s u a l l y
p r e s e n t a s i t s h e x a p h o s p h a t e e s t e r (P igm an, 1957). G a s - l i q u i d c h ro m a to g ra ­
p h y ru le d o u t th e p r e s e n c e o f I n o s i t o l , a n d p h o s p h o ru s w a s sh o w n to b e a b ­
s e n t b y a m o d if ie d .m e th o d o f F is k e a n d S u b b aro w (1925).
C i t r a t e w a s p r e s e n t in th e m y r o s in a s e p r e p a r a t io n (Figure 8); h o w - . .
e v er, e s t i m a t i o n s from th e g a s ch ro m a to g ra m s u g g e s t e d t h a t i t w o u ld m a k e ;.:
up o n ly a b o u t 1.5% o f th e t o t a l p r e p a r a t i o n . T here w a s a p o s s i b i l i t y t h a t th e
c i t r a t e p e a k o b t a i n e d r e p r e s e n t e d a m ore v o l a t i l e c i t r a t e d e r i v a t i v e a n d t h a t
a d d i t i o n a l c i t r a t e m ay h a v e b e e n r e t a i n e d b y th e c o l u m n . P rio r to f r e e z e -
'
d ry in g th e m y r o s in a s e s o lu tio n w a s d i a l y z e d a g a i n s t d i s t i l l e d w a te r to r e ­
m ove c i t r a t e io n s from th e pH 6.1 s o l u t i o n . , At t h i s pH l y s i n e (pKag = 10,5)
a n d a r g in in e (pKag = 12.5) w o u ld p o s s e s s p o s i t i v e l y c h a r g e d g ro u p s (-NHg+)
w h ic h c o u ld s e r v e to h o ld io n s s u c h a s c i t r a t e . W ith t h i s in m i n d , a c a l c u ­
l a t i o n b a s e d o n l y s i n e a n d a r g in in e c o n t e n t in th e m y r o s in a s e p r e p a r a t io n ,
w a s p e rfo rm ed to s e e how m u ch c i t r a t e c o u ld p o s s i b l y h a v e b e e n bound
b y t h e s e g ro u p s in a 1:1 m o la r a s s o c i a t i o n o f -N H g+ a n d c i t r a t e . T his /
32c a l c u l a t i o n s u g g e s t e d t h a t 8.2% o f th e
enzyme
p r e p a r a t io n c o u ld h a v e b e e n
c itra te .
The h ig h a s p a r t i c a n d g lu ta m ic a c i d c o n te n t o f m y r o s in a s e o ffere d
m a n y fre e c a r b o x y la te g ro u p s c a p a b l e o f b in d in g m e ta l io n s . In a d d itio n ,
s i a l i c a n d c i t r i c a c i d r e s i d u e s w h ic h w e re p r e s e n t w o u ld b in d c a t i o n s . So­
dium io n w a s th e m o s t p r e v a l e n t m e ta l io n e n c o u n te r e d b y t h e e n zy m e d u rin g .
p u r i f i c a t i o n . C a l c u l a t i o n s w e re m ade to s e e w h a t p e r c e n t o f th e m y r o s in a s e
p r e p a r a t io n c o u ld b e d u e to th e p r e s e n c e of s o d iu m . T h e s e c a l c u l a t i o n s a s ­
s u m e d t h a t s o d iu m w a s b o u n d by e a c h fr e e c a r b o x y la te g ro u p o f a s p a r t i c ,
g lu ta m ic a n d s i a l i c a c i d s k n o w n to be p r e s e n t a n d of c i t r i c a c id .w h ic h w a s
p r o b a b ly p r e s e n t , T a b le II in c l u d e s t h e s e r e s u l t s .
M o le c u la r W e ig h t o f M y r o s i n a s e
S e p a r a tio n of m o le c u le s b y g e l f i l t r a t i o n i s b e l i e v e d to r e s u l t from
d i f f e r e n c e s in s i z e of th e v a r io u s m o le c u le s a n d th e i r r e s t r i c t e d d iff u s io n
in to th e g e l p o r e s . M o l e c u l e s a p p e a r in th e e fflu e n t in o rd e r o f d e c r e a s i n g
s i z e . A lth o u g h m o le c u la r s i z e i s n o t n e c e s s a r i l y a good a p p ro x im a tio n for
m o le c u la r w e i g h t , m o le c u la r s i z e a n d w e ig h t a re c l o s e l y r e l a t e d for a s e r i e s
o f s im ila r m a c r o m o le c u le s ; e .g ., a s e r i e s o f p r o te in s or p o ly s a c c h a r id e s .
(G ra n a th a n d F l o d i n , 1961). ■As a r e s u l t g e l f il tr a tio n h a s b e e n a c c e p t e d a s a
c o m p a r a tiv e m e th o d for e s ti m a ti n g m o le c u la r w e ig h t (Andrews, 1964; P r i s t o u p i l , 1965; I w a ts u b o a n d C u r d e l , 1963; W h ita k e r, 1963; W ie l a n d e t a,I . ,1963).
In t h i s m e th o d a w o rk in g c u rv e w a s p r e p a r e d b y p lo ttin g r e l a t i v e m ig ra tio n
r a t e s o f s ta n d a r d c o m p o u n d s th ro u g h a g e l f il tr a tio n m edium v e r s u s th e ir
33
m o le c u la r w e i g h t s . U n know n m o le c u la r w e ig h ts w e re t h e n e s t i m a t e d o n th e
b a s i s of t h e i r r e l a t i v e m ig r a tio n r a t e s .
A 4 1 x 2 .5 cm B io -G e l P - 3 0 0 co lu m n w a s p re p a re d u s in g 0.05 M. sodium
c i t r a t e b uffer, pH 6.1, a s th e e lu tin g a g e n t . T h r e e - m i l l i l i t e r s o lu tio n s of th e
fo llo w in g m a t e r i a l s in c i t r a t e b u ffe r s e r v e d a s s t a n d a r d s : Blue D e x tra n 2000
(P harm acia F in e C h e m ic a ls , I n c , ) , b e t a - a m y l a s e , b e e f l i v e r c a t a l a s e , g l u c o s e
o x i d a s e , y e a s t h e x o k i n a s e a n d m u s c le p h o s p h o r y la s e (a ll from N u tritio n a l
B io c h e m ic a ls C o rp o ra tio n ). T h e s e s o l u t i o n s c o n ta i n e d a p p ro x im a te ly . 10 mg
o f s o lu te , e x c e p t in th e c a s e of p h o s p h o r y la s e w h ic h p ro v e d q u ite in s o lu b le
in c i t r a t e buffer. T h e s e s ta n d a r d s o l u t i o n s w e re p l a c e d o n th e B io -G e l c o l ­
umn a n d e lu t e d in 2-ml a l i q u o t s a t a r a t e o f 8 m l/h r . F iv e ,m illig ra m s of m y r o s i n a s e d i s s o l v e d in 3 ml o f c i t r a t e b u ffe r w e re t r e a t e d in a s im ila r m anner.
Blue D e x tra n 2000 (a p p ro x im a te m o le c q la r w e ig h t o f 21x10^) w a s no t
r e t a r d e d b y B io -G e l P - 3 0 0 a n d w a s a c o n v e n i e n t r e f e r e n c e s t a n d a r d . ' The:
r a t e o f p r o te in m ig r a tio n 'th r o u g h th e c o lu m n w a s d e term in e d ; r e l a t i v e to Blue
D e x tr a n 2000 a n d e x p r e s s e d a s R g g w h e re ; .
Rr t -X = E lu tio n v o lu m e o f Blue D e x tra n 2000
E lu tio n v o lu m e o f p ro te in
The e lu tio n v o lu m e w a s d e f in e d a s t h a t v o lu m e of e lu tin g a g e n t r e q u ire d to
r e a c h th e m id p o in t o f th e e lu t e d p e a k .
F ig u re 9 s h o w s th e R g g v e r s u s m o le c u la r w e ig h t c a l i b r a t i o n cu rv e
u s e d in t h i s w o rk . A s e c o n d co lu m n o f B io -G e l P -3 0 0 ( 3 8 x 1.6 cm) u s in g
0.5-ml s a m p l e s in th e s a m e b u ffe r a s b e fo re a n d e lu te d in 5-ml a liq u o ts , g a v e
e s s e n t i a l l y th e s a m e c a l i b r a t i o n c u r v e .
34
M y ro sin ase
2) C a t a l a s e
G lu c o s e O x i d a s e O
P -A m y la s e
O P h o s p h o r y la s e
H e x o k in a s e
/6 -A m y la s e
200
M o le c u la r W e ig h t (x 10" 3)
F ig u re 9 . Rb d v s . m o le c u la r w e ig h t for B io -G e l P -3 0 0 (4 1 x 2 .5 cm) e q u i l i ­
b r a te d w ith 0.05 M c i t r a t e buffer, pH 6.1.
.35 .
A m o le c u la r w e ig h t o f 320,000
by th is m e th o d .
±
15,OPQ w a s eptifiniatec} fpj; iT a y rp s i^ s e
.
■
■P r o n a s e D i g e s t i o n o f M y r p s i n a s e
I s o l a t i o n o f a fra g m e n t from m y r p s in a s e th a t c o n t a i n e d c a r b o h y d ra te
lin k e d to a s h o rt p e p tid e c h a i n w o u ld 'f u r th e r -gul?S t^ h tia tP th e c o v a le n t l i n k ­
a g e o f c a r b o h y d r a te to p r o t e i n . S p e c u la t io n ap to w h a t am in o a c i d w a s in v o lv e d in th e lin k a g e w o u ld a l s o be p o s s i b l e . In a d d i t i o n , th e num ber of
d if f e r e n t c a r b o h y d r a te m o ie tie s p r e s e n t a n d th e i r a p p ro x im a te s i z e c o u ld £>e
d e t e r m i n e d . A l o w - s p e c i f i c i t y p r o t e a s e p r e p a r a t io n from S tre p to m y c e s
'
g r i s e u s (p ro n a s e ) h a s fo u n d u s e in d e g r a d a tio n of g ly c o p r o te in s to g ly c p ^
p e p t i d e s w ith th e c a r b o h y d r a te m o ie tie s a p p a r e n t l y i n t a c t ' (T su g ita and A k ab o r i, 1959;. Nomoto e t W,., 1960; Eylar, 1962; Kamiyama a n d S c h m id , .1962;
F l e t c h e r e t a l . , 1963; J a m i e s o n , 1965; M o n tg o m e ry e t a l . , 1965; P a z u r e t
a l ., 1965).
P u rifie d m y r o s in a s e (135 mg) w a s s u s p e n d e d in 3 ml o f 0.05 M so d iu m
b o r a te buffer, pH 8.0, 0.015 M in G aC lg a n d w arm ed to 40QC . . O n e - t e n t h
m i l l i l i t e r o f 2% p r o n a s e (P ro te a s e ty p e VI,. Sigma C h e m ic a l C om pany) in th e
s a m e b u ffe r w a s w arm ed to 4 0 ° C , a d d e d to th e m y r o s in a s e s u s p e n s i o n a n d
i n c u b a t e d a t 4 0°C for 24 h o u r s . A nother 0.1 ml of p r o n a s e s o lu tio n w a s
a d d e d a n d th e in c u b a t io n re s u m e d for 137 h o u rs ., The i n c u b a t io n inixture
w a s c l e a r e d b y c e n t r i f u g a t i o n a n d I ml of s o d iu m b o ra te b u ffe r w a s u s e d to
w a s h th e r e s i d u e . T his s o lu tio n w a s c o m b in e d w ith th e o r ig i n a l s u p e r n a ta n t
so lu tio n .
36
The c o m b in e d s u p e r n a t a n t s o l u t i o n s from th e p r o n a s e d i g e s t i o n s (4 ml)
w e re a p p lie d to a S e p h a d e x G -2 5 co lu m n (4 4 x 1 .4 cm) e q u i l i b r a t e d w ith 0.1. N
a c e t i c a c i d a n d e l u t e d w ith th e s a m e s o l v e n t in 4-ml a l i q u o t s a t a r a te of
8 ml p e r hour. In lik e m anner, a b la n k s o lu tio n c o n ta i n in g 0.2 ml of th e p ro ­
n a s e s o lu tio n d i l u t e d to 4 ml w ith so d iu m b o r a te b u ffer w a s a p p lie d to th e
s a m e S e p h a d e x G -2 5 co lu m n a n d e l u t e d w ith 0.1 N a c e t i c a c i d . F ig u re s 10
a n d 11 sh o w t h e s e e l u tio n p a t t e r n s . The c a r b o h y d r a t e - c o n t a i n i n g p e a k s
from th e p r o n a s e - m y r o s i n a s e d i g e s t i o n w e re d e s i g n a t e d P M -2 5 -1 and
P M - 2 5 - 2 a n d w e re r e c o v e r e d by f r e e z e - d r y i n g f r a c tio n s 6 -9 a n d 10-15, r e ­
s p e c t i v e l y . In t h e p r o n a s e b la n k e l u t i o n , th e s a m e f r a c t i o n s w e re d e s i g ­
n a t e d P - 2 5 -1 a n d P - 2 5 - 2 , r e s p e c t i v e l y .
(This s e p a r a t i o n a n d s u b s e q u e n t
s e p a r a t i o n ^ w h ic h a re d e s c r i b e d b e lo w a r e s c h e m a t i z e d in F ig u re 17.)
F u rth e r f r a c t i o n a t i o n of th e c a r b o h y d r a te p r e s e n t in P M - 2 5 - 1 ,
P M - 2 5 - 2 , P - 2 5 -1 a n d P -2 5 -2 w a s a c c o m p l i s h e d by s u s p e n d in g e a c h in 0.1 N
a c e t i c a c i d , c e n tr if u g in g to rem ove m inor a m o u n ts of i n s o l u b l e s a n d s u b ­
j e c t i n g th e s u p e r n a t a n t s o l u t i o n s to g e l f i l t r a t i o n o n S e p h a d e x G - 75 (6 0 x 1.6
cm). P M -2 5 -1 a n d P - 2 5 -1 w e re a p p l i e d a s 2-ml s a m p le s (in c lu d in g a I - ml
0.1 N a c e t i c a c i d r e s i d u e w a s h in e a c h c a s e ) a n d c o l l e c t e d in 3-ml a l i q u o t s .
P M - 2 5 - 2 a n d - P - 2 5 - 2 w e re a p p lie d a s 3-ml s a m p l e s (in c lu d in g 0.1 N a c e t i c
a c i d w a s h p o rtio n s ) a n d c o l l e c t e d in 3-ml a l i q u o t s . The Oolumn flow r a te in
e v e r y c a s e w a s 11 ml p e r hour. F ig u r e s 1 2 -1 5 sh o w t h e s e r e s u l t s . It w a s
a p p a r e n t t h a t o n ly p a r t i a l s e p a r a t i o n o f th e c a r b o h y d ra te f r a c t i o n s w a s
a c h i e v e d b y t h e p r e v io u s g e l f i l t r a t i o n th ro u g h S e p h a d e x G -2 5 .
C a r b o h y d r a t e - c o n t a i n i n g p e a k s from b o th P M -2 5 -1 a n d P M - 2 5 - 2 w ere
37
PM'25-]
P M - 2 5-2
F r a c tio n Number
F ig u re 10. G e l f i l t r a t i o n o f th e p r o n a s e - m y r o s i n a s e d i g e s t o n S e p h a d e x
G -2 5 . ---- - T o ta l c a r b o h y d r a te ;
O. D. ggQ mjJi •
38
P -2 5 -1
P -25-2
F r a c tio n Number
F ig u re 11. G e l f i l t r a t i o n of th e p r o n a s e b la n k o n S e p h a d e x G - 2 5 .
------- T o tal c a r b o h y d r a t e ; --------O. D .2 go
d e s i g n a t e d P M - 7 5 - 1 , P M - 7 5 - 2 a n d P M - 7 5 - 3 . They w e re r e c o v e r e d by
f r e e z e - d r y i n g f r a c t i o n s 1 1-13, 3 2 -3 6 a n d 3 8 -4 2 , r e s p e c t i v e l y .
P M -7 5 -1 w a s t a k e n w ith o u t fu rth e r a tte m p ts a t p u r if ic a tio n a n d a n a ­
l y z e d for am in o a c i d a n d m o n o s a c c h a r id e c o n t e n t . A p o rtio n of P M -75-1
w h ic h w a s d i s s o l v e d in 0.1 N a c e t i c a c i d w a s e lu te d from S e p h a d e x G-100
(4 0 x 2 .5 cm) a s a s in g le , s lig h t l y - r e t a r d e d p e a k . P o rtio n s o f P M -7 5 -2 a n d
P M - 7 5 - 3 w e re d i s s o l v e d s e p a r a t e l y in I ml 0.1 N a c e t i c a c i d a n d p u rifie d by
g e l f i l t r a t i o n o n S e p h a d e x G-15 (48 x 1.6 cm). The e f f lu e n t w a s c o l l e c t e d in
2 -ml a l i q u o t s a t a r a t e of 8 ml p e r hour. T h e s e r e s u l t s a re sh o w n in Figure
16. The c a r b o h y d r a te w a s r e c o v e r e d by f r e e z e - d r y i n g a n d d e s i g n a t e d a s
PM -15-1 ( f ra c tio n s 17-23 from P M - 7 5 - 2 ) a n d a s P M -15-2 ( f ra c tio n s 3 0 -3 4
from P M - 7 5 - 3 ) .
39
P M -7 5 -1
P M -7 5 -2
F r a c tio n N um ber
F ig u re 12. G e l f i l t r a t i o n o f P M -2 5 -1 o n S e p h a d e x G -7 5 . ------ T o tal c a r b o ­
h y d ra te ; — O. D .2 80 mp*
40
o 0.2
F r a c tio n N um ber
F ig u re 13. G e l f i l t r a t i o n o f P - 2 5 -1 o n S e p h a d e x G - 75. —— T o ta l c a r b o h y ­
d r a te ; -----O. D .2 qo rnju-
F r a c tio n N um ber
F ig u re 14. G e l f i l t r a t i o n o f P -2 5-2 o n S e p h a d e x G - 75. —— T o ta l c a r b o h y ­
d r a te ; — O. D. 280 mpr
41
P M -7 5 -1
PM -75-3
-■S
0.8
PM -75-2
T
10
I
i
20
1
i
1
30
I
40
1
>
50
F r a c tio n Number
F ig u re 15.
d r a te ;
G el f i l t r a t i o n of P M - 2 5 - 2 o n S e p h a d e x G - 7 5 . -----T otal c a r b o h y ­
O. D .2 go mjw"
42
PM -15-1
P M -15-2
F r a c tio n N um ber
F ig u re 16. G e l f i l t r a t i o n of P M - 7 5 - 2 (A) a n d P M - 7 5 - 3 (B) o n S e p h a d e x
G-15. ----- T o ta l c a r b o h y d r a t e ; -----O. D . 2Rn
•
43
P r o n a s e - M y r o s i n a s e D i g e s t (PM)
or
P r o n a s e Blank (P)
v
S e p h a d e x G -2 5
(Figure 10 or 11)
S
I
P M -2 5 -2
or
P -2 5 -2
P M -2 5 -1
or
P -25-1
>f
S e p h a d e x G - 75
(Figure 14 or 15)
S e p h a d e x G - 75
(Figure 12 or 13)
V
P M - 7 5-2
X
P M - 75-1
Z
I
P M -7 5 -2
V
S e p h a d e x G-15
(Figure 16)
I
P M -7 5 -3
V
Sephadex G -1 5 I
(Figure 16)
I
PM -15-2
F ig u re 17. S ch em e for f r a c tio n a tio n b y g e l f i l tr a tio n of th e c a rb o h y d ra te
c o m p o n e n ts in th e p r o n a s e - m y r o s i n a s e d i g e s t o r in th e p r o n a s e b l a n k .
44
P M - 75-1, PM -15-1 a n d P M -1 5 -2 w e re a n a l y z e d for g a rb p h y d ra te c o n ­
t e n t b y g a s - l i q u i d c h ro m a to g ra p h y (F ig u re s 18-2 0). T h e s e r e s u l t s a re t a b u ­
l a t e d in T a b le V. T a b le VI g iv e s th e r e s u l t s o f a n am ino a c i d a n a l y s i s of
th e r e s u l t s o f a n am in o a c i d a n a l y s i s of P M - 75-1 a n d PM 1- I S - I fo llo w in g a
. 2 4 - h o u r h y d r o l y s i s . The S e p h a d e x G-15 co lu m n w a s s t a n d a r d i z e d by fo llo w ­
ing th e e l u t i o n of m o n o -, d i - a n d t r i s a c c h a r i d e s . T h is in fo rm a tio n su g g e s te d .' .
t h a t c a r b o h y d r a te in P M -1 5 -2 c o n s i s t s of m o n o s a c c h a r id e s . P a p e r c h ro m a ­
to g r a p h y s h o w e d g l u c o s e to b e th e o n ly C D - 1-p o s i t i v e c o m p o n e n t .
T a b le V.. C a r b o h y d ra te C o m p o s itio n of th e P r o n a s e - M y r o s i n a s e D ig e s t
F ra c tio n s.
.
G ram s of s u g a r /1 0 0 grants c a r b o h y d r a t e 3
S u g ar
P M -7 5 -lb
P M -IS -Ic
L -A ra b in o se
56.7
, . .
L -Fucose
• • •
. 0.9 •
D -X y io s e
4.2
3.9
D -M annose
• • •
17.6
D -G a lac to se
34.7
• • •
4.4
'24.1
D -G lu c o se
3.3'
D - G lu c o s a m in e ^
S ialic a c id e
• • •
L-^Ascorbic a c i d
T o ta ls
.
'
Present
P r e s e n t (?)
■ 50.4
...
100.0
P M -1 5 -2
100,2
P r e s e n t (?)
• • . •
a O b t a i n e d by g a s - l i q u i d c h ro m a to g ra p h y a s d e s c r i b e d in t e x t .
^ P r e p a ra ­
ti o n c o n t a i n e d '46.5% am in o a c i d . c - P re p a ra tio n c o n t a i n e d 15.2% am ino a c i d ,
d- A n a ly ze d , a s th e N - a c e t y l d e r i v a t i v e . e N - a c e t y l - n e u r a m i n i c a c i d .
M in u te s
Fig u re 18. G a s ch ro m a to g ra m o f th e t r i m e t h y l s i l y l d e r i v a t i v e s from th e 2 4 - h o u r P M -7 5 -1 h y d ro ly z a t e . (I) M e th y l c t - L - a r a b i n o s i d e ; (2) M e th y l OL-D - x y l o s i d e ; (3) M e th y l y S -D -x y lo s id e ;
(4) M e th y l (X - D - g a la c to s id e ; (5) M e th y l £ > -D -g a la c to s id e (6) M e th y l CL-D - g l u c o s i d e ; (7) M eth y l
/fl-D -g lu c o sid e .
M in u te s
Fig u re 19. G a s ch ro m a to g ra m of th e tr i m e t h y l s i l y l d e r iv a tiv e s from th e 2 4 - h o u r PM-15-1 h y d ro ly z a t e . (I) M e th y l L - f u c o s id e ; (2) M e th y l a - D - x y l o s i d e ;
(3) M e th y l /3 - D - x y lo s id e ; (4) M e th y l
a - D - m a n n o s i d e ; (5) M e th y l 2 - a c e t a m id o - 2 - d e o x y - O C- D - g lu c o s i d e ; (6) M e th y l a - D - g l u c o s i d e ;
(7) M e th y l / 5 - D - g l u c o s i d e ; (8) N - a c e t y l - n e u r a m i n i c a c i d .
M in u te s
Figure 20. G a s c h ro m a to g ra m o f th e tr i m e t h y l s i l y l d e r i v a t i v e s from th e 2 4 -h o u r PM -15-2 h y d ro ly z a t e . (I) P o s s i b l y m e th y l 2 - a c e t a m i d o - 2 - d e o x y - a . - D - g l u c o s i d e ; (2) M e th y l a - D - g l u c o s i d e ;
(3) M e th y l /6 - D - g l u c o s i d e ; (4) P o s s i b l y L - a s c o rb ie a c id ; o th e r s n o t id e n tif ie d or s u g g e s t e d .
48
T a b le VI. Amino A cid C o m p o s itio n of th e P r o h a s e - M y r o s i n a s e D ig e s t
F ra c tio n s. a
Amino A cid
Amino Acyl R e s id u e
(m g/100 mg sam p le)
P M -75-1
P M -^5-1
A s p a rtic a c i d
T h re o n in e
S e rin e
G lu ta m ic a c i d
P ro lin e
G ly c in e
A la n in e
V aline
Iso le u cin e
L e u c in e
T y ro sin e
P h e n y la la n in e
O rn ith in e
L y s in e
H istid in e
A rginine
6.6
2.2
2.8
6.4
1.5
1.6
3.7
. 2.5
1.5
4.8
2.2
1.8
0.8
1.9
1.2 '
5.0
T o ta ls
46.5-
G lu c o s a m in e
'
Amino Acid
|m m o le s/1 0 0 mg sam ple)
P M -7 5 -1 . . P M -1 5 -1
2,6
. 57.4
■ . • 21.6
■1.2
1.3
31.4
. 2.4
..... 49.5
0.8
. 15.5
2 7.2
1.5
.1.2
51.8
0.9
■ ' 25.2
■
0.4 „
.. .. 13.0
42.1
.0.3
0.2 ■
. 1 3 .8
0.2 .
12.5
• "
0.3
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' 8.2
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.6.9
. ; , 32.0
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. 12.2
14.9
18.4
. 8.3
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.
.
.
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.
' 17.4 ■
8.8
' 3.8 .
2 .7
.
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14.7
a B a s e d o n s i n g le s a m p l e s h y d r o ly z e d for 24 h o u r s .
The .d is c r e p a n c y in t h e .a m o u n ts of som e s u g a r s . ( p a r tic u la r ly .!>
.
a r a b i n o s e a n d D - m a n n o s e ) , w h ic h w e re p r e s e n t in m y ro s iria s e b e fo re a n d
a f t e r p r o n a s e d i g e s t i o n , s u g g e s t e d t h a t the- p ro n a s q m ay h a v e c o n ta i n e d
; .
s u g a r - m o d if y in g e n z y m e s , s u c h a s e p i m e r a s e s , o x i d a s e s , a n d d e c a r b o x y l­
a s e s . P r o n a s e w a s c h e c k e d for th e p r e s e n c e of s u c h e n z y m e s b y in c u b a tin g
i t w ith in d iv id u a l s o l u t i o n s (6-10 m g /m l) o f D - g lu c o s e , D -m a n n o s e , D g a l a c t o s e , D - g lu c o s a m in e , N - a c e t y l - n e u r a m i n i c a c i d a n d D - g a ^ a c tu r o n ic .
49
a c i d for 48 h o u rs u n d e r th e c o n d itio n s u s e d in p r e v io u s prbna.se in c u b a tio n s .
. A n a ly s e s u s in g p a p e r c h ro m a to g ra p h y s h o w e d no m o d if ic a tio n o f a n y of
th e s e su g ars by p ro n a s e .
• D is so c ia tio n of M y ro sin ase
M y r o s i n a s e i s o l a t e d d u rin g t h i s i n v e s t i g a t i o n a p p e a r e d to e x i s t a s 9
la r g e m o le c u la r a g g r e g a t e . T h is p ro m p ted a p a tte m p t to d i s s o c i a t e t h i s a g ­
g r e g a te in to s u b u n its ... S u ch a n e x p e r i m e n t , if s u c q e s s f y l , m ig h t lib e r a te
a d s o r b e d m a te r ia l ( p a r tic u la r ly c a r b o h y d r a te in t h i s c a s e ) b y d e s tr o y in g th e
s p a t i a l a r ra n g e m e n t n e c e s s a r y for a d s o r p tio n or e n c a p s u l a t i o n of th e c a r b o ­
h y d ra te . If th e C a rb o h y d ra te m o ie tie s p r e s e n t w e re c o v a l e n t l y lin k e d to th e
p o ly p e p tid e , c h a i n , t h e y w o u ld fo llo w p a r t i c u l a r p ro te in s u b u n i t s during
f r a c t i o n a t i o n a n d , t h e r e f o r e , be p a r t i a l l y . c h a r a c t e r i z e d . IJrea i s o n e of
s e v e r a l r e a g e n t s c a p a b l e p f d i s s o c i a t i n g m an y p r o te in s in s o lu tio n a n d w a s
u s e d in th e p r e s e n t i n v e s t i g a t i o n . The a f f e c t o f u rea o n p r o te in s i s n o t u n ­
d e r s t o o d , a lth o u g h m o s t t h e o r i e s a tte m p tin g a n e x p la n a tio n p o s t u l a t e t h a t
u re a c o m p e te s s u c c e s s f u l l y for s i t e s n e c e s s a r y for h y d ro g e n b o n d in g w ith in
a p r o te in a g g r e g a t e . T h is id e a a p p e a r s to be a p p l i c a b l e in m an y i n s t a n c e s ;
h o w e v e r, i t d o e s n o t e x p l a i n a l l p h e n o m en a o b s e r v e d in s t u d i e s . o n a s s o c i ­
a tio n a n d d i s s o c i a t i o n of p r o te in s (R e ith e l, 1963).
T en m illig ra m s o f p u r if ie d m y ro s iria s e w ere d i s s o l v e d in 2 ml o f 4 M
u re a s o lu tio n a n d e q u i l i b r a t e d 24 h o u rs a t 5 ° C . The s o lu tio n w a s a llo w e d to
r e a c h room te m p e ra tu re , a p p l i e d to a. S e p h a d e x G -2 0 0 (5 8 x 1.6 cm) column,
e q u i l i b r a t e d w ith 0.05 M so d iu m c i t r a t e buffer, pH 6.1, a n d e l u t e d w ith th e
s a m e b u ffe r in 5-ml a l i q u o t s a t a r a te o f 15 ml p e r hour. A s e c o n d e lu tio n o n
50
t h e s a m e co lu m n w a s ru n in th e sam e m a n n er u s in g a s o lu tio n o f p u rifie d
m y r o s in a s e in 8 M u re a (5 m g /m l). S im ila r e l u tio n c u r v e s w e re o b ta in e d
from th e tw o e l u t i o n s . The l a t t e r e l u tio n c u rv e i s sh o w n in F ig u re 2 1 .
F r a c tio n Number
F ig u re 2 1 . G e l f i l t r a t i o n o f a n 8 M u re a s o lu tio n of m y r o s in a s e o n S e p h a d e x
G -2 00 ( 5 8 x 1.6 cm). The s a m p le w a s e l u te d w ith 0.05 M c i t r a t e buffer,
pH 6.1, a t a flow r a t e o f 15 m l / h r in 5 -m l f r a c t i o n s .
In som e c a s e s p r o te in s u b u n its a re k n o w n to r e u n ite w h e n d ilu te d or
d i a l y z e d fre e of u re a . T h is c o u ld h a v e h a p p e n e d during th e e l u tio n w ith
c i t r a t e b u ffe r a b o v e . To m in im ize th e a s s o c i a t i o n of s u b u n its du rin g e lu ­
t i o n , a new S e p h a d e x G -2 0 0 colum n ( 4 6 x 1.6 cm) w h ic h u s e d 8 M urea a s
t h e e lu tin g a g e n t w a s p r e p a r e d . F iv e m illig ra m s o f p u rif ie d m y ro s in a s e
w e re d i s s o l v e d in 1.5 ml o f 8 M u r e a , e q u i l i b r a t e d a t 5°C for 2 4 h o u rs a n d
a p p lie d to th e S e p h a d e x G - 2 0 0 co lu m n a t room t e m p e r a t u r e . T w o - m illilit e r
51
f r a c t i o n s w e re c o l l e c t e d a t a flo w r a te o f 8 m l/h o u r. E a c h f r a c tio n w a s a n a ­
l y z e d for p r o te in a n d for c a r b o h y d r a t e , The r e s u l t i n g e l u tio n d ia g ra m is
sh o w n in F ig u re 22 . Three g ro u p s o f f r a c t i o n s w e re r e c o v e r e d from t h i s e l u ­
t i o n a n d t h e y w e re e x h a u s t i v e l y d i a l y z e d a g a i n s t so d iu m c i t r a t e buffer, pH
6.1. T hey w e re d e s i g n a t e d U - I ( f ra c tio n s 13-16), U - 2 ( f ra c tio n s 2 5 a n d 2 6),
U - 3 ( f ra c tio n s 29 a n d 30). I n c u b a tio n o f p o r tio n s o f t h e s e s o l u t i o n s a lo n e ,
o r in c o m b in a tio n , w ith s in ig r in s o lu tio n a t 3 7 ° C s h o w e d n e i t h e r t h io g lu c o s i d a s e n o r s u l f a t a s e a c ti v ity . G a s - l i q u i d c h ro m a to g ra p h y of i t s c a r b o r
h y d r a te s h o w e d U - I to h a v e a s u g a r c o m p o s itio n , s im ila r to th e c a r b o h y d ra te
o f th e o r ig i n a l m y r o s in a s e p re p a ra tio n , a n d U -2 a n d U - 3 g a v e e v id e n c e for
m in o r a m o u n ts of g l u c o s e a n d g a l a c t o s e .
52
F r a c tio n Number
F ig u re 22 . G e l f il tr a tio n of a n 8 M u re a s o lu tio n of m y r o s in a s e o n S e p h a d e x G -2 0 0 (47 x 1.6 cm). The s a m p le w a s e lu t e d w ith 8 M u r e a , a t a flow
ra te o f 8 m l / h r in 2 -m l f r a c t i o n s . ------- C a r b o h y d ra te by p h e n o l - s u l f u r i c
a c i d m e t h o d ; -----O p t i c a l d e n s i t y a t 280 mjj.
DISCUSSION
E x tr a c tio n a n d - P u r if ic a tio n o f M y r o s i n a s e
R e s u l t s o f t h i s r e s e a r c h a p p e a r to s u b s t a n t i a t e th e tw o - e n z y m e c h a r ­
a c t e r o f t h e m y r o s in a s e s y s t e m .. A lthough a c t u a l s e p a r a t i o n o f th e tw o e n ­
z y m e s w a s n o t a c c o m p l i s h e d , th e s p e c i f i c a c t i v i t y m e a s u r e m e n ts during
p u r if ic a tio n sh o w a d e f in ite v a r ia n c e of t h i o g l u c o s i d a s e a c t i v i t y from s u l f a t a s e a c ti v ity . It a p p e a r e d t h a t th e t h i o g l u c o s i d a s e a c t i v i t y w a s more s t a b l e
to w a r d s th e c o n d itio n s e m p lo y e d d u rin g p u r i f i c a t i o n . The s p e c i f i c a c t i v i t y
r a t i o o f th e t h i o g l u c o s i d a s e to s u l f a t a s e i n c r e a s e d from a n i n i t i a l 0.64 to
1.40 a f te r t h e fin a l p u r i f i c a t i o n s t e p .
T h e s e r e s u l t s d i s p u t e th e th e o ry of a, o n e - e n z y m e s y s te m p r e v io u s ly
r e p o r te d (N a g a s h im a a n d U c h i y a m a , 1959a; 1959b) w h ic h w a s p a r tia lly
b a s e d o n th e a p p a r e n t 1:1 r a tio b e tw e e n th e s p e c i f i c a c t i v i t i e s o f th e tw o
e n z y m e s u n d e r v a r io u s c o n d i t i o n s . N a g a s h im a a n d U c h iy am a (1959b) i s o ­
l a t e d m y r o s in a s e from w h ite m u s ta r d s e e d b y w a te r e x t r a c t i o n fo llo w e d by
e th a n o l p r e c i p i t a t i o n . E x a m in a tio n of T a b le I r e v e a l s g s im ila r ity in th e
s p e c i f i c a c t i v i t i e s o f th e tw o e n z y m e s th ro u g h th e e th a p o l p r e c i p i t a t i o n
s t e p ; h o w e v er, w ith a d d e d e n zy m e p u r i f i c a t i o n d i s c r e p a n c i e s in th e a c t i v i ­
t i e s r e s u l t e d . P re lim in a ry s t u d i e s o n th e i s o l a t i o n a n d p u r if ic a tio n o f my­
r o s i n a s e a p p e a r e d to s u g g e s t a s i n g l e - e n z y m e s y s t e m . In l a t e r e x p e r im e n ts
th e s in ig r in c o n c e n t r a t i o n w a s i n c r e a s e d for th e a s s a y i n c u b a t i o n s and
y ie ld e d th e r e s u l t s o f T a b le I . N a g a s h im a a n d U chiyam a (1959b) u s e d a n
e n z y m e : s u b s t r a t e r a tio la r g e r th a n t h a t u s e d in th e p r e s e n t w ork a n d , a s a
r e s u l t m ay n o t h a v e s a t u r a t e d th e e h z y jn e s ' a c t i v e s i t e s d u rin g th e a s s a y
54
p e r i o d . In th e p r e s e n t i n v e s t i g a t i o n m o s t p u r if ic a tio n s t e p s w e re perfo rm ed
a t , o r n ear, room t e m p e r a t u r e . T h is m a y h a v e c a u s e d a p r e f e r e n tia l i n a c t i ­
v a t i o n o f th e s u l f a t a s e . I n a c t i v a t i o n m ay n o t h a v e o c c u r r e d u n d e r c o ld room
c o n d itio n s a n d t h e c h a n g e in s u l f a t a s e a c t i v i t y m ay no t h a v e b e e n n o t i c e d .
F a ilu r e to s e p a r a t e th e t h i o g l u c o s i d a s e from th e s u l f a t a s e a llo w s room
for s p e c u l a t i o n . The p u r if ic a tio n p ro c e d u re o f G a in e s a n d G o e rin g (1960)
p e rfo rm ed th e e th a n o l p r e c i p i t a t i o n s t e p b e fo re th e am m onium s u lf a te p re ­
c i p i t a t i o n . In th e p r e s e n t i n v e s t i g a t i o n t h e s e s t e p s w e re r e v e r s e d a n d , in
som e m anner, m ay h a v e p r e v e n te d th e e n s u in g s e p a r a t i o n o n D E A E -ce llu lo se .
A p r iv a te c o m m u n ic a tio n from a n o th e r la b o r a to r y (C a ld e r o n , 1965) r e l a t e d
f a ilu r e to s e p a r a t e th e e n z y m e s by th e m e th o d of G a in e s a n d G o e r i n g . H o w ­
ev er, c o m m e rc ia l p r e p a r a t io n s of D E A E -ce llu lo se . v a ry ip t h e i r i o n - e x c h a n g e
p r o p e r tie s (P e te rs o n a n d Sober, 1962) a n d c o u ld a c c o u n t for s u c c e s s f u l f r a c ­
t i o n a t i o n in o n e i n s t a n c e a n d no f r a c t i o n a t i o n in a n o th e r.
The h ig h c a r b o h y d r a te c o n te n t in m y r o s in a s e a s i s o l a t e d in th e p re ­
s e n t w ork m ay be th e k e y to th e p ro b lem of s e p a r a t i o n . P o l y s a c c h a r i d e s
w ith t h e i r m a n y p o la r g ro u p s w ould h a v e g r e a t a f f in ity for th e p o la r p ro te in
m o l e c u l e s . T h erefo re, it i s c o n c e i v a b l e t h a t , if th e two e n z y m e s w ere a s ­
s o c i a t e d w ith a com m on c a r b o h y d r a te m o ie ty in th e i n t a c t s e e d , t h i s a s s o ­
c i a t i o n m ay be d if f ic u l t to d i s r u p t . T h e s e e n z y m e s m u s t a c t s i m u lta n e o u s ly
o r a t l e a s t s e q u e n t i a l l y w h e n c a t a l y z i n g t h e h y d r o ly s is o f s in ig r in u n d e r in
v iv o c o n d i t i o n s . Q u ite p o s s i b l y t h e s e e n z y m e s a re h e ld in a common v i c i ­
n ity b y a c a r b o h y d r a te m a trix a n d th e i n t a c t a g g r e g a te m ig h t b e i s o l a b l e .
D i s s o c i a t i o n s t u d i e s o n p u rif ie d m y r o s i n a s e in u re a s o l u t i o n sh o w e d m a rk e d
m o d if ic a tio n o f th e e n z y m e 's e lu tio n from S e p h a d e x G -2 0 0 (F ig u re s 21 a n d
55
22). N o th in g c o n c l u s i v e c o u ld b e d ra w n from t h e d a ta o b t a i n e d ; h o w ev er,
t h e a p p a r e n t l i b e r a t i o n of p o l y s a c c h a r i d e a n d th e h e t e r o g e n e i t y o f th e p ro ­
t e i n w h ic h w a s p r e s e n t s u g g e s t e d f r a g m e n ta tio n o f a p r o te in - c a r b o h y d r a te
c o m p le x b y u r e a . P r o n a s e s t u d i e s d i s c u s s e d l a t e r s u p p o rte d t h i s i d e a .
The s e d i m e n t a t i o n p a t t e r n of p u rif ie d m y r o s in a s e (Figure 2B) i n d i c a t e d
a p r e p a r a t io n of h ig h p u rity , b u t s h o w e d a s l i g h t s p l i t t i n g o f th e m ig ra tin g
b a n d . T h is r u le d o u t th e p o s s i b i l i t y of a h o m o g e n e o u s p r e p a r a t i o n . D is k
e l e c t r o p h o r e s i s a p p e a r e d to e x p la i n th e s p l i t t i n g in th e s e d im e n ta tio n p a t ­
t e r n . T h is t e c h n i q u e u t i l i z e s th e p r o p e r tie s of g e l f il tr a tio n in a d d itio n to
th e u s u a l e l e c t r o p h o r e s i s p a r a m e t e r s . The fiv e e n z y m a tic a lly a c t i v e b a n d s
r e s o l v e d b y e l e c t r o p h o r e s i s a p p e a r e d to c o n s t i t u t e a l l o f t h e p ro te in a p p lie d
to th e g e l . T h erefo re, th e s e d im e n ta tio n p a t t e r n of m y r o s in a s e n e e d not be
;
i n t e r p r e t e d a s s h o w in g im p u rity in th e e n zy m e p r e p a r a t i o n , b u t a s a s lig h t
s e p a r a t i o n of v a r io u s m y r o s i n a s e s p r e s e n t . .(E m phasis s h o u ld b e m ade of
t h e f a c t t h a t th e t e c h n i q u e for i s o l a t i n g a n d a s s a y i n g th e p r o te in b a n d s from
th e p o ly a c r y la m id e g e l w a s c ru d e a n d c o n ta m in a tio n b e t w e e n b a n d s c o u ld
h a v e o c c u r r e d .) The m u lti- e n z y m e p r e p a r a t io n w h ic h i s s u g g e s t e d by d is k
e l e c t r o p h o r e s i s i s in lin e w ith r e c e n t p r o t e i n , p a r t i c u l a r l y en zy m e, r e s e a r c h .
R e c e n t s t u d i e s h a v e i n d i c a t e d t h a t o n e o rg a n is m m ay p ro d u c e s e v e r a l d if f e r ­
e n t e n z y m e s w ith s im ila r s p e c i f i c i t y . About 30 e n z y m e s h a v e b e e n sh o w n to
e x i s t a s is o z y m e s (Gregory, 1961).. A d d itio n a l r e s e a r c h o n s p e c if ic ity , s p e ­
c i f i c a c ti v ity , a c t i v e s ite , in h i b i t i o n , e tc . w o u ld b e n e c e s s a r y b efo re a c c e p ti n g a n is o z y m ic n a tu re for m y r o s i n a s e . C a rb o h y d ra te w a s not fo llo w e d
\|
j
I:
d u rin g d is k e l e c t r o p h o r e s i s .
(I
56
C h e m ic a l C o m p o s itio n o f M y r d s i n a s e
The c h e m i c a l n a tu re o f m y r o s in a s e w h e n r e p o r te d a s a s in g le en zy m e
s y s te m h a s n e v e r b e e n d i s c u s s e d . K now ledge of th e c h e m i c a l n a tu re of my­
r o s i n a s e c o u ld p o s s i b l y e l u c i d a t e som e o f th e c o n f lic tin g r e p o r ts c o n c e rn in g
i t s p r o p e r tie s . Such in fo rm a tio n , w h e n u s e d in c o n ju n c t io n w ith m o le c u la r
w e ig h t a n d p r o t e o l y s i s s t u d i e s , m ig h t a l s o y ie ld fu rth e r in fo rm a tio n a s to
th e s i z e a n d num ber o f c a r b o h y d r a te m o i e t i e s k now n to b e p r e s e n t .
M a t e r i a l b a l a n c e w a s n o t a c h i e v e d by th e a n a l y s e s ru n o n p u rifie d
m y r o s in a s e in t h i s w o rk . A p p ro x im a te ly 91% of th e p r e p a r a t io n c o u ld be a c ­
c o u n te d for. P r o te in a n d c a r b o h y d ra te w e re th e m ajo r c o m p o n e n ts . x O n th e
b a s i s of th e v a r io u s t y p e s of c o m p o u n d s sh o w n to be a b s e n t , it a p p e a r e d
le g i t i m a t e to c o n s i d e r m y r o s in a s e in s o l u t i o n a s a p r o te in - c a r b o h y d r a te
c o m p le x c o n ta i n in g 19.2% c a r b o h y d r a t e . .O ther m a te r ia ls e x p e c t e d to be p r e ­
s e n t in th e f r e e z e - d r i e d m a te r ia l w e re w a te r of h y d ra tio n a n d c o u n t e r i o n s .
In th e p r e s e n t c a s e t h e s e c o u n te r io n s w o u ld of n e c e s s i t y in c lu d e p rim a rily
c i t r a t e a n d so d iu m i o n s . The c i t r a t e io n i s la rg e en o u g h to m ak e a s i g n i f i ­
c a n t c o n tr ib u tio n , so its c o n t e n t w a s e s t i m a t e d a s p r e v io u s ly d e s c r i b e d . So­
dium io n w a s e s t i m a t e d o n t h e b a s i s of th e a s p a r t i c , g lu ta m ic , s i a l i c a n d
c i t r i c a c i d c o n t e n t . • Sodium io n n e e d not b e th e o n ly c a t i o n p r e s e n t s in c e
o th e r c a t i o n s w e re p r e s e n t d u rin g v a r io u s s t e p s in th e p u r i f i c a t i o n .
I t w a s d if f ic u l t to s u g g e s t o th e r g e n e r a l ty p e s o f c o m p o u n d s th a t
c o u ld p o s s i b l y be p r e s e n t . The f a c t t h a t a 9 7% r e c o v e r y of n itr o g e n w a s o b ­
t a i n e d in th e am in o a c i d a n a l y s i s a n d t h a t s i a l i c a c id a n d g lu c o s a m in e w e re
a l s o p r e s e n t ru le d o u t m o s t o th e r c o m p o u n d s com m only fo u n d a s s o c i a t e d
w ith p r o t e i n .
57
In com m on w ith o th e r c a r b o h y d r a s e s (P azur e t a l . , 1963), m y r o s in a s e
w a s sh o w n to c o n t a i n la r g e a m o u n ts of a s p a r t i c a n d g lu ta m ic a c i d a n d o n ly
s m a ll a m o u n ts of s u l f u r - c o n t a i n i n g am in o a c i d s . The s i g n i f i c a n c e of t h i s
f a c t i s n o t k n o w n . In m a n y g ly c o p r o te in s th e d ic a r b o x y lic a m in o a c i d s a p ­
p e a r to b e th e am ino a c i d s w h ic h lin k c a r b o h y d ra te to t h e p r o t e i n . W h e th e r
t h e c a r b o h y d r a te is a t t a c h e d to p r o te in th ro u g h a c o v a l e n t l i n k a g e or sim p ly
b y a d s o r p t i o n , th e fre e c a r b o x y l g ro u p s o f t h e d ic a r b o x y lic a c i d s w o u ld c e r ­
t a i n l y p la y a n im p o rta n t r o le in s t a b i l i z i n g th e p r o te in - c a r b o h y d r a te c o m p le x
fo u n d in m a n y c a r b o h y d r a s e s . C o n v e r s e ly , th e low c o n te n t o f s u lfu rc o n ta i n in g am ino a c i d s m ay p o in t o u t t h a t th e c r o s s - l i n k i n g o f p e p tid e '
c h a i n s th ro u g h s u lfu r a to m s w a s n o t im p o rta n t for s t a b i l i z i n g th e s tru c tu re ,
a n d th e re fo re , th e a c t i v i t y o f t h e s e e n z y m e s . Such s p e c u l a t i o n is s u p p o rte d
b y th e e v id e n c e t h a t m an y e n z y m e s a re u n s t a b l e w h en s e p a r a t e d fro m .th e ir
n o rm a l a m o u n ts o f c a r b o h y d r a te (F is c h e r a n d - S te in , 1960).
A n a ly s is o f s u g a r r e s i d u e s b y g a s - d iq u id c h ro m a to g ra p h y in t h i s work
a p p e a r e d to be s a t i s f a c t o r y for n e u tr a l s u g a r s . A n a ly s e s for g lu c o s a m in e .
a n d s i a l i c a c i d w e re q u e s t i o n a b l e . E x te n d e d h y d r o ly s is in m e th a n o lle H C l
a p p e a r e d to d e s t r o y or m o d ify b o th of t h e s e c o m p o u n d s . G lu c o s a m in e form s
d e r i v a t i v e s w ith two d if f e r e n t r e t e n t i o n tim e s a n d one is a p p a r e n t l y l e s s
s t a b l e t h a n th e o th e r, • The u n s t a b l e p e a k i s sh o w n in F ig u re 6 w h ile th e
more s t a b l e p e a k n o rm a lly m a s k e d b y n e u t r a l s u g a r s b e g a n to a p p e a r in
F ig u re 8. The s i a l i c a c i d p e a k d i s a p p e a r e d a f te r a 4 8 -h o u r h y d r o l y s i s .
S i a l i c - a c i d ( N - a c e t y l - n e u r a m i n i c a c id ) c o n t a i n s m a n n o s a m i h e . As th e s i a l i c
a c i d c o n t e n t d i s a p p e a r e d , th e m a n n o s e c o n t e n t of m y r o s in a s e i n c r e a s e d
(Table IV).
T h is s u g g e s t s th a t o n e of th e d e g r a d a tio n p r o d u c ts from
.
58
N - a c e t y l - n e u r a m i n i c a c i d w a s m a n n o se , or m a n n o s a m in e w a s form ed a n d h a d
a r e t e n t i o n tim e s im ila r to m a n n o s e u n d e r th e c o n d itio n s u s e d in th e p r e s e n t
i n v e s t i g a t i o n . C i t r a t e io n g a v e th r e e g a s ch ro m a to g ra m p e a k s w h e n t r e a t e d
b y th e p r o c e d u re o u tlin e d p re v io u s ly . The m o s t in te n s e - p e a k w a s th a t sh o w n
in F ig u r e s 6, 7 a n d 8. A m uch l e s s i n t e n s e c i t r a t e p e a k w a s th e sam e a s t h a t
o f t h e (X -D -x y lo s e d e r i v a t i v e . The in t e r f e r e n c e of t h e , c i t r a t e p e a k w ith th e
x y l o s e p e a k i s sh o w n in F ig u re 8. X y lo s e c o n t e n t a s c a l c u l a t e d from th e 72^h o u r h y d r o ly s is w a s n o t i n c lu d e d in c a l c u l a t i n g th e a v e r a g e x y l o s e c o n te n t
o f m y r o s i n a s e . A th ir d c i t r a t e p e a k w a s u s u a l l y l e s s i n t e n s e t h a n th e f ir s t
tw o m e n tio n e d a n d o c c u r s j u s t p rio r to t h e p $ a k for L - f u c o s e .
M o l e c u l a r W e ig h t o f M y r o s i n a s e
A m o le c u la r w e ig h t o f 32 0,000 (± 15,000) w a s e s t i m a t e d for m y ro s in a s e
b y g e l f i l t r a t i o n t e c h n i q u e s . T h is te c h n i q u e a s s u m e d a s im ila r s h a p e a n d
d e g r e e o f s o l v a t i o n for e a c h p r o te in i n c lu d e d in th e s tu d y . T h ere is no r e a ­
s o n w hy t h e s e a s s u m p t i o n s s h o u ld b e tru e a n d , therefore,, t h e y probably, w e re
t h e m a jo r s o u r c e s fo r error. A h ig h d e g re e o f s o lv a t io n w o u ld i n c r e a s e th e
a p p a r e n t v o lu m e o f a p r o te in a n d r e d u c e i t s a b i l i t y to d if f u s e in to th e g e l
p o r e s . D if f u s io n of a m o le c u le is p ro b a b ly r e l a t e d to i t s s h a p e . - An a tte m p t
w a s m ade to g e t s t a n d a r d s w h ic h m ig h t a p p ro x im a te th e n a tu re of m y ro s in ­
a s e . T h ree o f t h e fiv e s ta n d a r d s u s e d for s t a n d a r d i z a t i o n o f t h e B io -G e l
P - 3 0 0 co lu m n w e re k n o w n to c o n t a i n c a r b o h y d r a t e .
In th e s e r i e s o f e l u t i o n s w h ic h w e re ru n to d e te rm in e t h e
v a l u e s for
m y r o s in a s e a n d t h e s ta n d a r d p r o te in s , a s i n g l e p e a k w a s o b t a i n e d o n ly in
t h e e l u t i o n p a t t e r n o f m y r o s in a s e a n d p h o s p h o r y l a s e . The m u l t i p l e - p e a k
e l u t i o n s w h ic h r e s u l t for t h e p ro te in s t a n d a r d s a p p e a r to b e c o n s i s t e n t w ith
59
p r e s e n t k n o w le d g e o f d i s s o c i a t i o n a n d a s s o c i a t i o n o f p r o te in (R e ith e l, 1963).
In e a c h c a s e , som e of th e p e a k s o b ta in e d a p p e a r to r e p r e s e n t p a r t i c l e s i z e s
w h ic h h a v e b e e n p r e v io u s ly r e p o r te d for t h a t p r o t e i n . B e t a - a m y l a s e h a s
b e e n sh o w n to e x i s t in fo u r p a r t i c l e s i z e s (E nari, 1965). C a t a l a s e h a s b e e n
re p o r te d to e x i s t in m an y s t a t e s o f a g g r e g a tio n (R e ith e l, 1963); th re e p a r­
t i c l e s i z e s p re d o m in a te d i n t h i s w ork: 248,000, 175,000, 62,000. H e x o k in a s e
w a s e l u t e d p re d o m in a te ly a s o n e m ajo r p e a k w h ic h c o r r e s p o n d e d to i t s a c ­
c e p t e d m o le c u la r w e i g h t . M u s c l e p h o s p h o r y l a s e c o n t a i n s i n a c t i v e s u b u n its
o f 125,000 m o le c u la r w e i g h t . The p h o s p h o r y la s e p r e p a r a t io n u s e d in th is
w ork w a s o n ly s l i g h t l y s o lu b le w h e n m ix e d w ith c i t r a t e b uffer. T his co u ld
a c c o u n t for th e s i n g le e l u t i o n p e a k o b t a i n e d . E lu tio n of g l u c o s e o x id a s e
y i e l d e d a p e a k c o n s i s t e n t w ith i t s r e p o r te d m o le c u la r w e ig h t o f 150,000
( P a z u r ^ t a l . , 1965). H o w ev er, t h i s c o m p o n e n t w a s n o t th e m a jo r in g r e d ie n t
in th e e n zy m e p r e p a r a t i o n .
The p o s s i b i l i t y of c o n ta m in a ti o n in t h e v a r io u s p ro te in s ta n d a r d s w a s
■
r e a l . M o le c u la r w e ig h ts o f v a r io u s p e a k s in e a c h p ro te in s t a n d a r d a s e s t i ­
m a te d from t h e p r e s e n t s ta n d a r d c u rv e (Figure 9) c o r r e l a t e d w e ll w ith m o le ­
c u l a r w e ig h ts p r e v io u s ly r e p o r t e d . Such r e s u l t s a p p e a r e d to j u s t i f y a n e s t i ­
m a tio n of th e m o le c u la r w e ig h t of m y r o s in a s e in t h i s m anner.
P r o n a s e a n d D i s s o c i a t i o n S tu d ie s o h M y r o s i n a s e
D i g e s t i o n o f m y r o s in a s e w ith p r o n a s e g a v e a t l e a s t th r e e d i s t i n c t
c a r b o h y d r a t e - c o n t a i n i n g f r a c t i o n s a f te r a s e r i e s of f r a c t i o n a t i o n s by g e l f i l ­
t r a t i o n (Figure 17). C o n ta m in a tin g c a r b o h y d r a te from p r o n a s e a p p e a r e d
s m a l l . T h is w a s sh o w n b y g e l f i l t r a t i o n o f a p r o n a s e b la n k in th e sam e .
60
m a n n e r a s th e p r o n a s e - m y r o s i n a s e d i g e s t (F ig u res 12-15). The l a r g e s t c a r - .
b o h y d ra te fra g m e n t ( P M - 75-1) w a s s l i g h t l y r e ta r d e d b y S e p h a d e x G-100
w h ic h w o u l d . i n d i c a t e a m o le c u la r w e ig h t o f a p p r o x im a te ly 100,000. P M - 75-1
w a s sh o w n to c o n t a i n 46% a m in o a c i d l e a v i n g 54% a s c a r b o h y d r a t e .. A t l e a s t
th r e e p o s s i b l e s t r u c t u r e s a re s u g g e s t e d : (I) th e c a r b o h y d r a te m ay be a
s e r i e s of o l i g o s a c c h a r i d e m o i e t i e s lin k e d to a p o ly p e p tid e c h a i n , (2) th e
c a r b o h y d r a t e i s o n e m o ie ty lin k e d to a n e x t e n s i v e p o ly p e p tid e c h a i n , or (3)
t h e c a r b o h y d r a te i s a la rg e p o l y s a c c h a r i d e a s s o c i a t e d w ith m an y sh o rt p e p ­
t i d e g r o u p s . The n o n - s p e c i f i c p r o te o ly tic a c t i v i t y of prona s e a n d th e e x - .
te n d e d p e rio d o f i t s in c u b a t i o n w ith m y r o s in a s e w ould s e e m to ru le o u t (I)
a n d (2). In a d d i t i o n , d i s s o c i a t i o n o f th e m y r o s in a s e in 8 M u re a s o lu tio n
a p p e a r e d to fr e e t h i s c a r b o h y d r a t e m o ie ty o f m o s t p r o t e i n . ■Arab in o s e , x y r
lo se, g a l a c t o s e a n d g l u c o s e w e re th e m o n o s a c c h a r id e u n its p r e s e n t in
■P M - 7 5 -1 .. A ra b in o s e is n o t com m only p r e s e n t in g ly c o p r o te in (Spiro, 1963);
h o w e v e r, i t h a s b e e n r e p o r te d in ^ - g l u c o s i d a s e p r e p a r a t io n s (Ve'ibel, 1950)
a n d in a g l y c o p e p t id e i s o l a t e d from w h e a t flo u r (Kundig a n d N e u k o m , 1963).
\
X y lo se, g a l a c t o s e a n d g l u c o s e h a v e b e e n fo u n d In some e s t a b l i s h e d g ly c o ­
p r o te in s , b u t u s u a l l y in c o n ju n c t io n w ith h e x o s a m in e , s i a l i c a c i d a n d / o r f u c o s e .(Spiro, 1963; M a r le r a n d D a v id s o n , 1965). The p e r c e n t a r a b i n o s e in
P M - 75-1 w a s q u e s t i o n a b l e a n d s h o w e d no r e l a t i o n to th e a r a b i n o s e c o n te n t
o f i n t a c t m y r o s i n a s e . A lth o u g h th e am in o a c i d w a s not i d e n t i f i e d , g a s c h r o ­
m a to g ra p h y o f a s ta n d a r d am ino a c i d m ix tu re i n d i c a t e d t h a t a n amino a cid had.
. a r e t e n t i o n s im ila r to a r a b i n o s e o n a S E - 30 co lu m n u n d e r c o n d itio n s u s e d in
t h i s w o rk . M e th a n o lic H C l m ay no t h a v e h y d ro ly z e d t h i s a m in o a c i d free
Z
61'
from m y r o s in a s e ; h o w e v er, a f t e r p r o n a s e d i g e s t i o n it w a s p ro b a b ly fre e to
form a m e th y l e s t e r a n d tr i m e t h y l s i l y l d e r i v a t i v e .
PM -15-1 a p p e a r e d to be th e o th e r c a r b o h y d r a te m o ie ty p r e s e n t to a n y
e x t e n t in m y r o s i n a s e . S u g ar a n d am ino a c i d c o n te n t a lo n e s u g g e s t a g ly c o p e p tid e n a t u r e H e x o s a m i n e , f u c o s e , m a n n o se , s i a l i c a c i d a n d a s p a r t i c a c i d
a re com m on c o m p o n e n ts o f c o v a l e n t l y - l i n k e d c a r b o h y d ra te a n d p e p tid e (Gotts c h a l k , 1963; Spiro, 1963; M o n tre p il e t aj.., 1962). In som e c a s e s g lu ta m ic
a c i d (Li, 1963), s e r in e a n d th r e o n in e (Blix, 1963) h a v e b e e n fo u n d to s e rv e
a s th e lin k in g am ino a c i d s . The PM -15-1 f r a c tio n w a s p r e s e n t in both
P M -2 5 -1 a n d P M -2 5 -2 i n d ic a tin g it w a s r e t a r d e d by S e p h p d e x G -2 5
( P M - 7 5-2 in F ig u r e s 12 a n d 15). T h is s u g g e s t e d a m o le c u la r w e ig h t l e s s
t h a n 4000. E lu tio n from S e p h a d e x G - 75 p l a c e d PM -15-1 n e a r t h e lo w er m o le ­
c u l a r w e ig h t lim it (1000) for f r a c t i o n a t i o n o n t h i s g e l f i l t r a t i o n m ed iu m .
PM -15-1 w a s sh o w n to b e h e te r o g e n e o u s b y e l u tio n from a S e p h a d e x G-15
c o lu m n . T h is t r e a t m e n t , w h ic h a llo w e d f r a c tio n a tio n of m a t e r i a l s w ith mo­
l e c u l a r w e ig h ts l e s s t h a n 1500, s u g g e s t e d t h e p r e s e n c e of a t l e a s t th re e
c a r b o h y d r a te f r a c t i o n s . Amino a c i d a n a l y s i s s h o w e d PM -15-1 to c o n ta i n 15%
am in o a c i d . T h is w o u ld im p ly t h a t c a r b o h y d r a te m o ie tie s w h ic h w e re pre*s e n t m ay h a v e h a d a m o le c u la r w e ig h t ra n g e of 800-1200. R e s u l t s in c lu d e d
in T a b le V did n o t p r e d ic t m o le c u la r w e i g h t s o f th is s i z e . B a s e d on th e a s ­
s u m p tio n t h a t o n e r e s i d u e of fu c o s e w a s p r e s e n t p e r . c a r b o h y d r a te grouping
a m o le c u la r w e ig h t o f 16,000 w o u ld be e s t i m a t e d . U sin g x y l o s e or g l u c o s ­
a m in e a s th e b a s i c s u g a r r e s i d u e a m o le c u la r w e ig h t o f a t l e a s t 5000 w o u ld
b e e x p e c t e d . H o w ev er, t h e e v id e n c e t h a t PM -15-1 w a s h e te r o g e n e o u s p ro b ­
a b ly e x p l a i n e d t h i s a p p a r e n t d i s c r e p a n c y b y a llo w in g th e p o s s i b i l i t y t h a t
62
e a c h c a r b o h y d r a t e m o ie ty m ay h a v e h a d a d if f e r e n t s u g a r c o n t e n t . G a in e s
a n d G o e rin g (1960) s h o w e d a t pH 8.5 a n e le c tr o p h o r e ti c m o b ility o f 2 .5 8 8 x
10~5 cm2 v o l t " I s e c " ! for th e t h i o g l u c o s i d a s e a n d - 0 . 8 6 3 x 1 0 " ^ cm2 v o lt" !
s e c " I for th e s u l f a t a s e . H u o ta ri (1962) s h o w e d th e t h i o g l u c o s i d a s e p r e p a r a ­
ti o n to be fre e of s i a l i c a c i d . T h is e v id e n c e w h en c o r r e l a t e d w ith th e r e ­
s u l t s o f th e p r e s e n t w ork s u g g e s t e d a g ly c o p r o te in c h a r a c t e r for b o th e n ­
z y m e s p r e s e n t . S ia lic a c i d a t pH 8.5 w o u ld te n d to im p art a n e g a ti v e c h a r ­
a c t e r to p r o te ip d u rin g e l e c t r o p h o r e s i s . C a r b o h y d ra te m o i e t i e s c o n ta in in g
s i a l i c a c i d w ere, th e re fo re , p ro b a b ly r e c o v e r e d from th e s u l f a t a s e m o le c u le
p r e s e n t . ■A s s ig n m e n t o f t h e o t h e r s u g a r s p r e s e n t to a p a r t i c u l a r en zy m e w a s
no t p o s s i b l e from th e r e s u l t s o b t a i n e d . T h ere w ere u n e x p la i n e d d i f f e r e n c e s ,
in th e s u g a r c o n t e n t of i n t a c t m y r o s in a s e a n d s u g a r s r e c o v e r e d a f te r p r o n a s e
d ig e stio n .
The th ir d c a r b o h y d r a te f r a c tio n from p r o n a s e d i g e s t i o n a p p e a r e d to be
m o n o s a c c h a r i d e . R e la tiv e i n t e n s i t i e s o f th e c a r b o h y d ra te p e a k s in F ig u re s
12 a n d 15 s u g g e s t t h a t a t l e a s t 50% of th e c a r b o h y d ra te d e t e c t e d by th e
p h e n o l - s u l f u r i c a c i d m e th o d w a s e lu t e d as- P M - 7 5 -3 ( u ltim a te ly P M -1 5 -2 ). It
w o u ld b e c o n v e n i e n t to s u g g e s t p o s s i b l e c a r b o h y d r a s e a c t i v i t y in th e p ro n ­
a s e p r e p a r a t io n w h ic h w a s u s e d . H o w ev e r, th e g a s ch ro m a to g ra m from
P M -1 5 -2 (Figure 2 0) w a s no t t y p i c a l for th e s u g a r s k n ow n to b e p r e s e n t in
m y r o s i n a s e . P a p e r c h ro m a to g ra p h y s h o w e d o n ly g lu c o s e to b e p r e s e n t . T h is
g l u c o s e w a s p ro b a b ly e x t r a c t e d from th e C M - c e l l u l o s e a n d S e p h a d e x u s e d
d u rin g 'this w o r k . A sc o rb ic a c i d w a s t e n t a t i v e l y i d e n tif ie d a s o n e of th e g a s
c h ro m a to g ra p h y p e a k s . T h is is r e a s o n a b l e s in c e a s c o r b i c a c i d is know n to
be a n a c t i v a t o r for m y r o s in a s e (N a g a s h im a a n d U c h iy a m a , 1959a). The f a c t
63
t h a t th e m y ro s in a s e p r e p a r a t io n c o n t a i n e d o n e c a r b o h y d ra te m o ie ty w ith a n
a p p ro x im a te m o le c u la r w e ig h t o f 55,000 a n d o th e r m o ie tie s o f a p p ro x im a te ly
1000 m o le c u la r w e ig h t r u le d o u t th e p o s s i b i l i t y t h a t a m a jo r am o u n t of free
m o n o s a c c h a r i d e s w a s a l s o in th e p r e p a r a t i o n . If m y r o s in a s e h a s a m o le c u ­
l a r w e ig h t n e a r 320,000 a n d i s 19.2% c a r b o h y d ra te , th e c a r b o h y d r a te m o ie tie s
d e s c r i b e d w o u ld a c c o u n t for th e t o t a l c a r b o h y d r a te p r e s e n t . The num ber of
th e v a r io u s c a r b o h y d r a te c o m p o n e n ts in P M - 15 - 1 c a n n o t b e d e te r m in e d frpm
t h e p r e s e n t d a t a . The o rig in a n d n a tu re o f P M -1 5 -2 w a s n o t r e s o l v e d d u rin g
th is in v e stig a tio n .
SUMMARY
I.
M y r o s i n a s e h a s b e e n i s o l a t e d from s e e d s o f B r a s s ic a ju n c e a by th e
c o n s e c u t i v e s t e p s o f w a te r e x t r a c t i o n o f th e m ille d o i l - f r e e s e e d s , am m oni­
um s u l f a t e p r e c i p i t a t i o n , e th a n o l p r e c i p i t a t i o n , c e l l u l o s e io n - e x c h a n g e
c h ro m a to g ra p h y a n d g e l f i l t r a t i o n .
2 . The e n z y m e p r e p a r a t io n a p p e a r e d to c o n ta i n b o th a t h i o g l u c o s i d a s e
a n d a s u l f a t a s e ; h o w e v er, t h e tw o e n z y m e s w e re no t s e p a r a t e d . P u r if ic a tio n
t e c h n i q u e s w h ic h w e re u s e d y ie ld e d a 2 5 0 - f o ld i n c r e a s e in th e s p e c i f i c a c ­
t i v i t y for th e t h i o g l u c o s i d a s e , b u t o n ly a 115-fold i n c r e a s e for th e s u l f a t a s e .
3.
M y r o s i n a s e a s i s o l a t e d in t h i s i n v e s t i g a t i o n c o n t a i n e d 19.2% c a r b o ­
h y d r a t e . P r o t e o l y s i s in v o lv in g a - S tr e p to m y c e s g r is e u s p r o t e a s e a n d d i s s o ­
c i a t i o n in u re a s o lu tio n s u g g e s t e d t h a t t h e c a rb o h y d ra te w a s p r e s e n t a s a n
a s s o c i a t e d h i g h - m o l e c u l a r w e ig h t h e t e r o p o l y s a c c h a r i d e a n d a s s e v e r a l lo w m o le c u la r w e ig h t m o ie tie s w h ic h w e re p ro b a b ly bound c o v a l e n t l y to th e p ro ­
te in .
4.
The am ino a c i d a n d s u g a r c o n s t i t u e n t s of both th e i n t a c t m y r o s in a s e
a n d th e c a r b o h y d r a te f r a c t i o n s o b ta in e d b y p r o t e a s e a c t i o n . w e r e d e te r m in e d .
5.
The m o le c u la r w e ig h t of m y r o s in a s e w a s e s t i m a t e d by a g e l f i l t r a ­
ti o n m e t h o d .
6.
The s i g n i f i c a n c e of th e r e s u l t s o b t a i n e d w ere d i s c u s s e d in r e l a t i o n
to t h o s e p r e v io u s ly r e p o r te d .
LITERATURE. CITED
A ndrew s, P. (1964), B io c h e m . I. 9 1 , 222 .
B entley, R ., S w e e le y , C. C ., M a k ita , M ., W e ll s , W. W. (1963), B io c h e m .
B iophys. Res. C o m m u n . 1 1 , 14.
B e rn a rd !, G ., A p p e l l a , E ., a n d Zito, R. (1965), B io c h e m is try 4 , 1725.
B e tte lh e im ^ J e v o n s , F, R . (1958), A d v a n . P r o te in C h e m . 13 , 3 5 .
B lix , G . (1963).. A nn. N. Y. A c a d . S c i . 106 (Art. 2), 164.
B o lto n , C. H . , C lam p, J. R ., a n d H o u g h , L . (1965), B io c h e m . L 9 6 , 5 c .
B o u t r o n - C h a r l a n d , A . F ,, a n d R o b iq u e t, P. J. (1831), I. P h a r m . 17, 294; a s
l i s t e d b y G a in e s (1960).
Bussy,. A . (1840), I. P h a r m . 2 6 , 39; a s l i s t e d b y G a in e s (1960).
D a v is , B. T. ( 1 9 6 4 ) , A n n . N . Y . A c a d . S c i . 121 (Art. 2), 4 0 4 .
D o d g s o n , K . S. (1961), B io c h e m . I. 7 8 , 3 1 2 .
D u b o is , M .,■ G i l l e s, K . A ., H a m ilto n , J. K ., R ebers, P„ A ., a n d S m ith , F. (1956),
■A n a l . C hem . 2 8 , 3 5 0 .
E n a r i , T -M . (1965), C e r e a l S c i. T o d ay 1 0 , 5 9 4 .
'
E t tlin g er, M . G ., a n d L u n d e e n , A . J. (1956), T. A m. C h e m . Soc . 7 8 , 4 172.
E ttlin g e r, M . G ., a n d L u n d e e n , A . J, (1957), T. A m . C h e m . Soc . 7 9 , 1764.
Eylar, E . H . (1962), B io c h e m . B iophys . R es . C o m m u n . 8, 1 9 5 .
F au re, M . G . (1831).. I. P h a r m . 17, 299; a s l i s t e d b y G a in e s (1960).
F is c h e r , E. H . , and- S te in , E , A. (I960),. The E n zy m es 4, 3 0 1 .
F is k e , C. H ., a n d Subbarow , Y. (1925),. I. B i o l . C h e m . 6 6 , 3 7 5 .
F o l c h e , J., L e e s, M ., a n d S ta n le y , G. H. S. (1957), L B io l. C h e m . 2 2 6 , 497.
F le tc h e r , A. P., M a r k , G. S., M a r s h a l l , R. D., a n d N e u b e r g e r , A . (1963),
B iochem I. 87, 2 6 5 .
G a in e s , R . D . (1960), Ph. D.. T h e s i s , M o n ta n a S ta te C o l l e g e , B o ze m a n .
66
G a in e s , R. D., a n d G o e r i n g , K . J. (1960), B io c h e m . B iophys. Res. C o m m u n . 2 ,
207.
G a in e s , R. D., a n d G o e r i n g , K . J. (1962), Arch . B io c h e m . B iophys. 9 6 , 13.
G m e l i n , R ., a n d V ir ta n e n , A . I. (1959), Acta C h e m . S c a n d . 1 3 , 1 4 7 4 .
G o o d w in , T. W., a n d M o rto n , R. A. (1946),. B io c h e m . T. 4 0 , 62 8 .
G o rd o n , H . T., T h o r n b u rg , W ., a n d W e r u m , L. N . (1956), A n a l. C h e m , 2 8 ,
849.
■G o t t s c h a l k , A. (1963),.. A nn. N.Y. A c a d . S c i. 1 0 6 .(Art. 2), 168.
G r a n a t h , K. A ., a n d . F l o d i n , P. (1961), M a k r o m o l. C h e m . 4 8 , 16 0 .
G regory, K . F. (1961), A nn. N. Y. A c a d . S c i. 94 (Art. 3), 657.
H e d g le y , E . J., a n d O v e r e n d , W. G. (1960), C h e m . & I n d . (L o n d o n ), 3 7 8 .
H e l f e r i c h , B., Richter,-Wv, a n d G runler, S. (1938), Ber. V e rh a n d l. s a c h s . A k a d .
■W i s s . L e i p z i g , M a th - p h y s . K l . 8 9 , 385; a s l i s t e d in V e ib el (1950). .
H e l f e r i c h , B., (1943),. Ergeb. E n z y m f o r s c h . 9 , 70.
H u o t a r i , F„ (1962), M a s t e r ' s T h e s i s ,■ M o n ta n a S t a t e - C o l l e g e , B o z e m a n .
Iw atsubo,. M ., a n d C u r d e l , A . (1963), C o m p t. r e n d . 2 5 6 , 5 2 2 4 .
J a m i e s o n , G . A . (1965),. I. B io l. C h e m . 24.0, 2 9 1 4 .
K a g a n , J., a n d M abry, T. J. (1965),■ A n a l. C h e m . 37, 2 8 8 .
K a lo k a rf H . (1947), T. B io l. C h e m . 167, 4 6 1 .
K am iyam a , S., a n d -S c h m id , K. (1962), B io c h e m . B iophys. Acta 5 8 , 8 0 .
K e e n , J. L ., a n d O p ie , J. W. (1957), T a p p i 4 0 , 100; a s l i s t e d b y S m ith , F., .
a n d M on tg o m ery , R. (1959), C h e m is tr y of P la n t Gums a n d M u c i l a g e s ,
R h e in h o ld P u b li s h in g C o ., N ew Y o rk , p. 100. •
K u n d ig , W., a n d N e u k o m , N . (19 63), H elv . C him . Acta 4 6 , 1 4 2 3 .
L e v e n e , P.A. (1926), . H e x o s a m in e s a n d M u c o p r o te in s , Longm ans, G re e n a n d
C o ., L o n d o n .
Li, Y. T., (1963), D i s s e r t a t i o n A bstr. 2 4 , 1 3 7 4 .
67
Lowry, O. H
R o se b ro u g h , N . J.,- Farr, A„ L
T. B i o l . C h e m . 193 , 2 6 5 . •
a n d R a n d a ll, R. J. (1951),
M a rle r, E ., a n d D a v i d s o n , E . A. (19 65), Prop. N a t. A c a d . S c i . U. 8. 5 4 , 648 .
M ey e r, K . (1945),. A d v a n . P r o t e i n C h e m . 2 , 2 4 9 .
M o n tg o m ery , R ., Lee, Y. C., a n d -W u , Y. C. (1965), B io c h e m is try 4, 5 6 6 .
M o n t r e u i l , J., S p i k , G ., C h o s s o n , A.,- S e g a r d , E ., a n d -S c h e p p le r, N . (1962),
. E x tra it " C h ro m a to g ra p h ie — Sym posium II", S o c ie te Beige d e s S c ie n c e s
P h a r m a c e u t i q u e s , p . 47. =
M oore, S. a n d S te in , W. H . (1954), I. B io l. C h e m . 2 1 1 ,, 8 9 3 .
N a g a s h im a , Z., a n d U c h iy a ma, M . (1959a), B u ll. Agr. C h e m . Soc. Tapan 2 3 , ■
555.
N a g a s h im a , Z ., a n d U c h i y a m a ,-M . (195,9b), L Agr. C h e m , Soc. J a p a n 3 3 , 881;
a s l i s t e d in C h e m . A b s t r a c t s 57, 3 7 8 7 h .
N a g a s h im a , Z ., a n d U c h iy a m a , M . (1959c). I. Agr. C h e m . Soc. Tapan 3 3 , 980;
a s l i s t e d in. C h e m . A b s t r a c ts 57, 154981.
N a g a s h im a , Z ., a n d U c h i y a m a , M . (1959d), I. Agr. C h e m . Soc. I a p a n 3 3 , ,
1 144; a s l i s t e d in -C h e m . A b s t r a c ts 5 8 , 4 7 6 9 c .
N e u b e r g , C., a n d S c h o e n e b e c k , O. v. (1933), N a t u r w i s s e n s c h a f t e n 2 1 , 4 0 4 .
Nomoto, M ., N a r a h a s h i , Y., a n d M u ra k a m i, M . (1960), I. B io c h e m . (Tokyo)
48, 906.
O h k u m a , S., a n d S h i n o h a r a , T„ (1963), Proc. J a p a n A c a d . 3 9 , 6 8 6 .
P a rtrid g e , S. M . (1 9 4 8 ),-Biochem I. 4 2 , 2 3 8 .
P azu r, J. H . , K leppe, K ., a n d B a ll, E . M . (1963),-A rc h . B io c h e m . Biophys. 103,
515. '
P a z u r, J. H . , K leppe, K ., a n d -C e p u re , A . (1965),-A rch. B io ch em . Biophys. I l l ,
351.
P e t e r s o n , E . A ., and- Sober, H . A. (1962),. M e th o d s E n z y m o l. 5, 6.
P ig m a n , W. (1957), The C a r b o h y d ra te s , A c ad e m ic P r e s s , In c ., N ew York,
P .2 7 1 .
P r i s t o u p i l , T. I. (1965), T -C h r o m a to g . 1 9 , 6 4 .
68
R e i t h e J 7 F. J. (1963),-A d v a n . P r o te in C hem . 1 8 , 1 23.
Sandberg, M
a n d H o lly , O. (1 9 3 2 ),-J. B io l. C h e m ■ 9 6 , 4 4 3 .
S c h m id , K. (1964), A b s t r a c ts , 147th N a tio n a l M e e tin g of th e A m erican
C h e m ic a l S o c ie ty ,. P h il a d e lp h ia , P a . , p . 3 6 .
S im k in , J. L,,- S kinner, E . R ., a n d -S e s h a d r i , H . S. (1964),. B io c h e m . T. 9 0 , 316
Spiro, R . G . (1963), N ew E n g l. T. M e d . 2 6 9 , 5 6 6 .
S ta c e y , M . (1946),. A d v a n . C a r b o h y d ra te C h e m . 2 , 161.
Sumner, J. B. (192 5), I. B io l. C h e m . 6 2 , 2 8 7 .
S w e e le y , C. C ., B entley, R ., M a k ita , M . , a n d W e lls , W. W. (1963), L Am.
C h e m . Soc. 85 , 2497.
S w e e le y , C- C ,, a n d W a lk e r, B. (1964), A n a l . C h e m . 3 6 / 1 461.
T s u g ita , A., a n d A k a b o r i, 8. (1 9 5 9 ),-1. B io c h em . (Tokyo) 4 6 , 6 9 5 .
V e i b e l , S. (1950),. The E n z y m e s I , 583 .
v o n Euler, H .,. a n d .E r ic k s o n , S t. E . (1926), F e rm e n tfo rs c h u n g 8 , 5 1 8 .
W h is tle r , R . L ., a n d B eM iller, J, N . (1962), M e th o d s - C a r b o h y d r a te C h e m .
395.
,
W h ita k e r, J. R. (1963),- A n a l. C h e m . 3 5 , 1 9 5 0 .
W h ite , T. (1940), I. C h e m . S o c :, 4 2 8 .
W i e l a n d , T., D u e s b e r g , P., a n d -D e t e r m a n n , H . (1963), B iochem . Z . 337, 303
Mo n t a n a
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