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Studies on cellulolytic enzymes
B I O C H I M I C A |-."r I,il'f'tP|IYSICA AC'I-A
9
BBA X 2 X 2 0
STUDI ES ON CELLULOLYTIC
ENZY MES
II. M U L T I P L I C I T Y O F T H E C E L L t . I L O L Y T I C E N Z Y M E S O F POL Y P O R U S V E R S t C O L O R G,. P E T T E R S S O N
^ND J P O R A T t t
lnstffvCe of 131ochemiMr),, University of I.:pp.~ala, l;ppsulu (Suarden) (R,*,'civt:d M a y z 5 t h . t~li2)
SItMMARY
I. F o u r cellulolytic c o m p o n e n t s h a v e been isolated from c u l t u r e filtrate of t'olylSorus versicolor. z. T h e m o l e c u l a r w e i g h t o f one o f t h e c o m p o n e n t s has been d e t e r m i n e d to be 5 I OflO.
3. A c o m p a r i s o n has bee.n m a d e of t h e a m i n o acid c o m p o s i t i o n s o f the 5x oooc o m p o n e n t a n d t h e p r e v i o u s l y isolated x, o o o - c o m p o n e n t . 4- A n e x p l a n a t i o n for t he o c c u r r e n c e o f m u l t i p l e e n z y m m o f v a r y i n g cellulol}tic a c t i v i t i e s has b ee n p r o p o s e d . lXr,~o,~ucrlox T h e c e l l u l o l y t i c e n z y m e s o f a large n u m b e r of fungi h a v e been purified using block e l e c t r o p h o r e s i s on p o t a t o s t a r c h as d e s c r i b e d b y MASD~I~ a M. t. T h e s e s t u d i e s h a v e s h o w n t h a t m o s t o f t h e fungi s t u d i e d t o daze a p p e a r t o p r o d u c e several cellulolytic c o m p o n e n t s . T h e e x i s t e n c e o f m u l t i p l e c o m p o n e n t s also has been d e m o n s t r a t e d by e l e c t r o p h o r e s i s o f o t h e r e n z y m e s y s t e m s . V a r i o u s e x p l a n a t i o n s h a v e been p r o p o s e d t o a c c o u n t for t h i s h e t e r o g e n e i t y z. F o r e x a m p l e , M I L L E R e t alfl r e c e n t l y h a v e s h o w n t h a t e l e c t r o p h o r e t i c a l l y s e p a r a t e d c o m p , , n e n t s differ in specific a c t i v i t y on c a r b o x y m e t h y l cellulose a n d cel l opent aos e . I t t h e r e f o r e a p p e a r s likely t h a t som e o f t h e cellul o l y tic c o m p o n e n t s m a y p l a y different roles in t h e d e g r a d a t i o n o f cellulose. T h e p r e s e n t w o r k was u n d e r t a k e n to e l u c i d a t e t h e m u l t i p l e - c o m p o n e n t celltdase s y s t e m ot Polyporus versicolor. I t is a cor, t i n u a t i o o o f t h e i n v e s t i g a t i o n r e p o r t e d in t h e p r e c e d i n g a r t i c l e ~. .~tETm)DS
AND IcEst:tA-S
Source of the enzyme T h e m a t e r i a l for t h e p r e s e n t e'~7~'iments wa_~ o b t a i n e d a_~ filtrates f r o m c u l t u r e s Gf t h e w o o d - d e s t r o y i n g b a s i d i o m y c e t e , Polyporus veTs/co/or, gr(,wn in a n t i n e r a l s a l t s m e d i u m w i t h cellulose as sole s our c e o f c a r b o n . T h e fi l t rat e w as c o n c e n t r a t e d a n d f r a c t i o n a t e d on S e p h a d e x as d e s c r i b e d emrlier t, g i v i n g 3 fractions, d e s i g n a t e d B, C a n d D. 13zochim. Hio,phys. Aaa. 67 0 0 ~ , 3 ) .o---J~
IO
G. PETTERSSON, J. PORATH
gnzym~ assays T h e cellulase a c t i v i t y o f all f r a c t i o n s w a s d e t e r m i n e d as t h e a m o u n t o f s o l u b l e reducing substances formed when the enzyme was incubated with cazboxymethyl cellulose or c e l l u l o ~ sr~l. D e t a i l s of t h e a n a l y s i s p r o c e d u r e s were g i v e n p r e v i o u s l y ' .
Zone el~dropkoresis P,rificatio~J of Fyaction B (s~nall scale) Z o n e e l e c t r o p h o r e s i s w a s p e r f o r m e d in a v e r t i c a l b e d s o f P e v i k o n p o w d e r ( P e v i k o n C 870 o b t a i n e d f r o m Fo:~fatbolaget, S t o c k h o l m (Sweden)). T h e m a t e r i a l w a s purified by extensiv-, washing with ethanol, then with 4 M hydrochloric acid and fina!Ly w i t h distilled w a t e r . T h e p o w d e r w a s t h e n s u s p e n d e d in 0.o 5 .~1~ p y r i d i n e a c e t a t e b u f f e r ( p H 4.5). T h e e l e c t r o p h o r e s i s c o l u m n , a z × 35 c m glass t u b e , w a s cln~.d a t t h e b o t t o m w i t h a p l a t e o f p o r o u s p l a s t i c ( V y o n a v a i l a b l e f r o m P o r o u s Plastic Ltd. Dagenham Docks, Essex (England)). The column was provided with a h o r i z o n t a l c a p i l l a r y side t u b e (z m m in d i a m e t e r ) z c m f r o m t h e b o t t o m . T u b i n g o f p o l y x d n y l c h l o r i d e 60 c m in lenh-th a n d z m m inside d i a m e t e r w a s c o n n e c t e d to t h e c a p i l l a r y side t u b e . T h ~ c o l u m n w a s p a c k e d w i t h g l a s s b e a d s ( B a n o t i n i N o . zz) t o a h e i g h t o f z m m a b o v e t h e c a p i l l a r y side a r m . P e v i k o n p a s t e f r e e d f r o m a i r b y e v a c u a t i o n w a s t r a n s f e r r e d to t h e c o l u m n t h r o u g h a p a c k i n g t u b e t o f o r m a 3 o - c m h i g h column. The f~tering properti~ of the bed were tested by passing a narrow zone of dinitrophenylethanolamine through the column.
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Fig. x. Zone eh.ctropherogram of Fraction B. Bed dirw.'nsiona ; × z8 era; buffer o.o5 M pyridineacetate (pH 4.5): currenL 9 mA; voltage. 700 V. Fracttor~q 7-Y4 (1~;) and 3o-36 (Bsl w~re pooled. × - - x , absorbency ~ zSom/~; C ) - ~ , ccllula.se actis'ity against earbo:~ymethyl cetlul~s~ (arbitrary units). Biochi,n.
Biophys÷ At:a,
67 (z963) 9--z 5
CELLULOLYTIC ENZYMES. I!
I!
z o m g o f F r a c t i o n B w e r e d i s s o l v e d in 0. 4 m l 0.05 ,~I p y r i d i n e - a c e t a t c b u f f e r { p H 4-5) a n d p l a c e d o n t o p o f t h e c o l u m n . T h e z o n e w a s a | l o w c d t o f i l t e r d o w n x c m a~xd t h e c o l u m n t h e n a r r a n g e d ;Ls d e s c r i b e d b y POI~ATtt et al. e. E a c h e l e c t r o d e v e s s e l c o n t a i n e d 4 1 o f b u f f e r (0.05 M p y r i d i n e - a c c t a t e ( p H 4-5))- T h e p o l y v i n y l c h l o r i d e t u b i n g w a s c o n n e c t e d w i t h a p e r i s t a l t i c p u m p v i a 0.2 × z o - c m l a t e x t u b i n g . T h e b u f f e r in t h e electro,de v e . ~ e l s w a s m i x e d t w i c e a t s u i t a b l e t i m e i n t e r v a l ~ t o k e e p t h e p H c o n s t a n t . Af':er e l e c t r o p h o r e s i s f o r z o h a t 7o0 V a n d 9 m A . c o n t i n u o u s e l u t i o n w a s b e g u n a t a c o n s t a n t flow r a t e o f 4.5 ml:;h. A f t e r a s e c o n d p e r i o d o f 2o h. electrophoresi.~ w a s s t o p p e d a n d t h e m a t e r i a l on t h e c o l m n n d i s p l a c e d w i t h f r e s h b u f f e r a t a flow r a t e o f x.5 m l / h . F r a c t i o n s c o n t a i n i n g 0.5 m l w e r e c o l l e c t e d . A f t e r d i l u t i o n to z.5 m l t h e a b s o r b a n c y a t z S o nap w a s m e a s u r e d in a B e c k m a n D U s p e c t r o p h o t n m e t e r . T h e r e s u l t s a r e s h o w n in F i g . z a n d r e v e a l t w o a c t i v e f r a c t i o n s : B t a n d I~,~'. T h e d i s t r i b u t i o n o f c d h t l a s c a c t i v i t y a n d a b s o r b a n c y wa~s f [ m n d t o c o i n c i d e o v e r t h e g r e a t e r p a r t o f t h e B~ z o n e . W i t h i n e x p e r i m e n t a l e r r o r , t h e c u r v e s fi~r a c t i v i t y on c e l l u l o s e sol a n d c a r l x ~ x y m e t h y l c e l l u l o ~ ,verc c o i n c i d e n t . T h e f r a c t i o n s w e r e po0Ev3 a s i n d i c a t e d in Fig. t a n d t h e n f r e e z e - d r i e d .
Purification of Fraction B (large sctde) L a r g e r q u a n t i t i e s o f F r a c t i t m B (5t~ m g / m l b u f f e r ) w e r e f r a c t i o n a t e d b y a ~imilar p r o c e d u r e . A ~- × 3 o - c m c o l u m n w a s u s e d . I t wa.~ p r o v i d e d w i t h a c o o l i n g j a c k e t o f methylpolymethacrylate (Perspex) but was not equipped for continuous elution. The e l e e t r o p h o r e s i s t i m e r e q u i r e d a t 400 V a n d 9.2 m A w a s c a l c u l a t e d f r o m t h e r e g u l t s o f e a r l i e r e x p e r i m e n t s . T h u s it w a s p o s s i b l e t o s t o p t h e e l e c t r o p h o r e s i s w h e n t h e f a s t moving component was near the bott,m of the column. After elution, the analysis o f t h e f r a c t i o n s c o l l e c t e d r e v e a l e d t h a t t h e mat~,riM d i s t r i b u t i o n w a s c o m p a r a b l e w i t h t h e e a r l i e r f i n d i n g ~ a s s h o w n in Fig. r.
Purification of Fraction C F r a c t i o n C a ! ~ wa~ f r a c t i t n i a t c d wn t h e ,.amt: c t d u m n a n d in e x a c t l y t h e ~ a m e m a n n e r a s F r a c t i o n B (amMl .~'.xle). T h e p l o t s o f a c t i v i t y a n d a b s o r b a n c y o f t h e v a r i o u s f r a c t i o n s a r e v e r y s i m i l a r t~J tho.-,e fi~r F r a c t i o n B, b u t t h e a c t i v i t y a n d a b sorbancy curves are not coincident. The fraction~ were pooled and freeze-dried as i n d i c a t e d in Fig. 2.
CarbvlLvdrate anal~'sis T h e h e x o ~ c o n t e n t o f c , , m p , m e n t B 1 w a s e s t i m a t e d t o b e z"g.. (w/w) b y t h e o r c i n o l m e t h o d a s d e s c r i b e d b y Vnss~.:uR 7 T h e h e x o s c c o n t e n t o f comp~)ao.nt D p r e v i o u s l y h a d b e e n d e t e r m i n ~ d t o b e 33~',o ( w / w ) I.
Amino acid anMysis " l ~ e a m i n o a c i d c o m p o s i t i o n o f o x i d i z e d .~araples o f c o m I x m e n t ~ Bt a n d D w e r e d e t e r m i n e d u s i n g a B e c k m a n - S p i n c o a u t o m a t i c a m i n o a c i d a n a l y z e r a n d tile p r o c e * T h e e v L z y m c s present in the fractions obt:Lined from g e t f i l l ~ a f i o n or elvctroph~rcsis r e f e r r e d t o u s i a g t h e s a m e labeLq a s t h e f r a c t i o n s ~ t , I l s e t ~ . w b e t h e x t h e y a x e p u x e o r n o t .
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F i g . ". Z o n e e l . ~ t r o p h e r o g r a m o f F r a c t i o n C, Bt~l d i m e n ~ o n s , buffex', c u r r e n t a n d v o l t a g e a s i n F i g . t. F r a c t i o n s G - t 4 [C1) a n t i a ~ - 3 1 (Ct) w e r e p o o l e d , X - - × , a b ~ o r b a n c y a t ~ g o m j u ; C>-- - - O , celtulase activity against carboxymethyl cellulose (arbitrary units).
TABI,E I'RFLIMINARY
AItIINO
A minn ~a4~t~
I
A.CID COMPOSITION
Samp~t Bt
OF COMPONKNTS
5a4,ttpt* D
Bit A N D
D
B,~o o/
ami4~ ~£~ (BJD)
I,ymne ! Li~tidine Ammonirt A r girtine C~-~teic acid Aspartic acid .Methionine suifone Threoniue Serine Glutamic acid Proline Glycmc Alaninc VoAine l~ol~ucine LeucLr.e Ty-rozi~e Phen~,[~Lsninc
0-2.57 0.079 o-944 o. [ 5 o o.o3o 0.832 0.o98 o. 4 9 3 o.5z6 o.49o 0.2 to o(,o9 o-4n5 0.265 o. t g 3 o.34I o. x 2 o o.2z 3
0.029 o.ot 5 0-395 o.o3o o.ol 7 o.x605 0.0o8 o. I o 5 x o.zo4z o. t o t o 0.0770 o-o(~¢3 0-0703 o.o744 0.0405 0-0726 Destroyed" 0.0288
* The oxidizing agent was d~,rtroyed using hydrobromi¢
9.0 5.3 z-4 5.o t .8 5.z 12 4- 7 5.0 4.9 2 7 8.9 5-9 3.6 4 +R 4-7 -7-7
a c i d t.
B i o c h i m . B i o p A l t s . ,.iota. 6 7 (196,3) 9 - - t 5
CELLULOLYTIC
ENZY-MI'~S. I I
I3
d u r e o f SPACKblAN d'~ al. s. B e c a u s e o f Lite s m a l l a m o u n t o f m a t e r i M avail;~ble, o n l y a p r e l i m i n a r y d e t e r m i n a t i o n of a m i n o acid c o n t e n t could be made. T h e samples c o n s i s t e d o f f r e e z e - d r i e d m a t e r i a l ~ 2 . 6 o m g o f c o m p o n e n t B , a n d x.2o m g o f c o m p o p,e n t D. T h e h y d r o l y s L g t i m e w a s 24 it. O n e t h i r d o f t h e m a t e r i a J wzLs p l a c e d o n e a c h c o l u m n o f t h e a n a l y z e r . T h e a m i n o a c i d c o m p o s i t i o n s o f t h e .~amplcs are s h o w n in T a b l e I.
Sedime~dation st,dies Sedimentation analysis was performed with a Spinco model E ultracentrifuge p r o v i d e d w i t h a m o d e l D a n a l y t i c a l r o t o r anti a .~ynthetic b o u n d a r y cell. T h e e x p e r i ments were carried out at an average speed of 5')78o rev./min andat a temperature o f 2o °. z m l o f a x~/o (w/v) s o | u t i o n in o.z ,%~ p y r i d i n e a c e t a t e b u f f e r ( p H 5.0) wa~ used. C o m p o n e n t B] g a v e a single s e d i m e n t a t i n g boundary.'. T h e s e d i m e n t a t i , m c o e f f i c i e n t sa0,~o w a s f o u n d t o be 4 . z I $. C o m p ~ m e n t B= w h e n e x a m i n e d u n d e r t h e .,mane c o n d i t i o n s al~o a p p e a r e d t o be homogenecm.~ a n d s~,,o e q u a l t o 4 . z 6 .% w a s o b tairled. 1.:, !: :.f m;-¢,wi;!l .nn,~:!:. ;t T]''C,--'~rcr'." t,~ r,lrl c o r n p r , n e n t C: ;II,'l (:2 iYt ~1 lOW c o n c e n t r a t i o n n a m e l y o . 3 ~ 0 [w,,v). U n d e r the~e ,~ut~,ptimal r o n d i t i o n s it w a s difficult t o d i s c o v e r a n y p o l y d i s p e r s i t y , sz~.~ wa~ f o u n d to be 2.o S for C t a n d r . 9 S h)r C~.
l)etermi~lalion of melecz,lar weight T h e m o l e c u k : r w e i g h t o f c o m i , o n e , t l~ 1 w a s d e t e r m i n e d b y t h e , n e t l , u / o f A R C , I ISAL,,'--". T h e ~.entrifuge cell w;L-~ filled t o a h e i g h t o f xo m m w i t h a t°A) (,.v/x.) .¢,olution tff t h e p r o t e i n .~r. o.1 M p y r i d i n e a c e t a t e ( p H 5-o). T h e a v e r a g e sp~'.ed o f r o t a t i o n w a s Ix 573 r e v . / m i u a t 2o ". T h e m o l e c u l a r w e i g h t e s t i m a t e s in Table. ! I w e r e d e t e r m i n e d "I:~ T]I.E [I M O L E C t T L A R %VF.[GHT Or-" L'~SLLUL.%SE C O M P O N F . N T ~1 F~qrl:.~.XTE[) s~*" 5 5 1 ) I . % l E N T A T I O N .%NAi%'.%IS
The vitriatton among the ~ a : u ~ is l,robabl.v due to u;l
m
]b z4 3-" 4o 48 56
.t,'4 ~.~J
5z 0oo 48 9o.> ..,%.f, Z.~O 5i 3cxl
64 72. 80 ~t~ 9 t'
Average
55 400 53 200
4,'~ 5o0 4 ° Cx.~o 51 2,.,o
51 q o o
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51 ~=oo
5 t (x>o
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50 8o0 B~ch*,,. L~iophy¢. Acta. 67
(t963) 9 - ' 5
14
G. P E T T E R S S O N . $, PORATH
from e x t r a p o l a t e d meniscus a n d b o t t o m values, assuming t -- t~ .- o.29. The value o.7x for fi was calculated from the a m i n o acid composition according to MCMEEKIN AND M A R S H A L L i t .
DISCUSS|ON
The rc_~ults of this investigation clearly demon.~trate the occurrence of multiple ccllulolytic enzymes in Polyporjts v~sicolor. T h e e n z y m e c o m p o n e n t s differ in moleculax size a n d differences in substrate specificity h a v e been o b ~ r v e d . In addition to /~-glucosidase, at I~ast 5 cellulolytic c o m p o n e n t s seem to be present in the culture fllt rate. Additional c o m p o n e n t s are indicated b y the p a t t e r n of distribution of a c t i v i t y in the various fractions (Figs. x. 2). We have recognized two pairs o f c o m p o n e n t s moving with slightly different relative velocities on Sephadex G 75 (see ref. 4) a n d with sedimentation coefficients o f 4.2 a n d 2.o S respectively. In a preceding paper 4 it fifth component, designated D, ha-~ been described. L a c k o f material has prevented examination of the p(~,~sible significance of the occurrence of paired enzymes. Only components B l and D have been obtained in quantities sufficient for s e m i q u a n t i t a t i v e a m i n o acid analysis a n d molecular weight determinations. Despite the u n c e r t a i n t y of a m i n o acid analysis, several points of evidence in the results appear w o r t h y of mention. C o m p o n e n t s B t a n d D contained conspicuous a m o u n t s of acidic a m i n o acids. The a m m o n i a c o n t e n t in the h y d r o l y s a t e was very high. Electrophoresis revealed t h a t B t was only slightly positively charged a t p H 4-5 while component D was immobile at this pH, The~se facts t o g e t h e r s t r o n g l y suggest t h a t a large n u m b e r of c a r b o x a m i d e side groups are present in b o t h ce$1ulase components. As sho~vn in Table 1, nine amino acids o u t of sixteen occur in a b o u t the same proportions. These findings suggest t h e existence of c o m m o n polypeptide units. A n u m b e r of cellulolytic organisms produce m u l t i - c o m p o n e n t cellula~e5 tt. I t has been shown t h a t the~e components occasionally differ in specificity on polysaccharides of various molecular sizes. Based on these observations STONE~* propo.~ed the following scheme for the degradation of cellulose. "oliga.~" Cellulose - - ~
cellr~,lextrin ~ - - ~ c e l l o b i u ~
"l~flyasc"
It is evident from the p r e ~ n t work t h a t the basidiomycete Polyporus t'ersicolor produced several cellulolytic components. T h e gel filtration m e t h o d , described in the preceding article 4, gave a fractionation according to molecular size; the low-molecularweight proteins being less mobile t h a n those o f high molecular weibht. These earlier results therefore show, t h a t the ~-glucosidase in F r a c t i o n A, is of higher molecu!~r weight t h a n t h e e n z y m e s pre.sent in Fractions B, C a n d D. T h e l a t t e r e n z y m ~ are active on cellulose and c a x b o x y m e t h y l cellulose. The a c t i v i t y curves for cellulose and c a r b o x y m e t h y l cellulose are displaced in relation to one a n o t h e r in a m a n n e r t h a t suggests t h x t the e n z y m ~ m o s t active on cellulose are o f larger molecular size t h a n those active on carbox.vmethyl cellulose. L a c k of coincidence between these two kinds of a c t i v i t y has been d~rnonstrated evvn more convincingly for cellulolytic enz y m e s of some other organisms (to be published later). These circumstances indicate Bioch~m. B i o p h y s . ,4eta. b 7 ( I 9 6 3 ) 9~-i5
CEI+I.III.fYI+'+'TIC E.~Z~'31E.¢,. II
T~
t h a t an inverse relationship m a y exist b e t w e e n the molecular size o f the e n z y m e c o m p o n e n t s a n d t h a t o f the substrates: L o w - m o l e c u l a r - w e i g h t c e I l u l a ~ s h o w i n g h i g h e s t a c t i v i t y on gluco~idic b o n d s in p o l y s a c c h a r i d e c h a i n s w h i m the h i g h - m o l e c u l a r w e i g h t e n z y m e s , w i t h the /~-gluco~ida,e as t h e mt~t e x t r e m e c~L-;e, preferenti'~lly c a t a l y z e t h e h y d r o l y s i s of oligosaccharictes. %ucL a molecular size r e l a t i o n s h i p s h o u l d facilitate the d e g r a d a t i o n of cellulo.,~ b y ;t geries of e n z y m e s a c t i n g in a se~!uential m a n n e r . To test the v a l i d i t y of this h v p o t h e s i s , t h e ~ i n v e s t i g a t i o n s will be e x t e n d e d t o include cellula_ges from o t h e r fungi, t o g e t h e r w i t h a kinetic characteriz a t i o n o f the c o m l ~ m e n t s i.¢,olated. A C K NO',VI. I-~lit ;E M 16 N'[ S
W e are i n d e b t e d to P r o f e ~ o r g.. "I~sEJAt,~; a n d Dr. E. B. COWLing; fi~r their kind interest. For skilful technica! assistance we wi~h to t h a n k Miss I~. INGEL+MAN. This i n v e s t i g a t i o n wa~ s u p p o r t e d b y a grant from the S w e d i s h N a t u r a l Science l',esear+-I-. Council and from the N a t i o n a l Scien,t. F u u n d a t i o n (G I87O2). REF'ET~ ENCE5 t M+ M ^ I ~ D E L S . G . I.. MlUt.EK a . ~ O Ft. V,'. S t . A r ~ r . J ~ . . :lr¢t,t. t~och,.'m. Riopk.,.'s. 9 3 It0,',1) It F, ~'RODI..X.WSKI, ,-J~tn. P,'.I/, .-lead. Net.. w-! (/,:~,)i! ¢,'.,.-. | G . L . MILLIe.n &Nil It{. BI~,ZG.~.LIS, Al'ch. ~Jiocktt'Jt ~ff*o#ll',.$., 95 (IOG/) | 9 -
It5+
• G, ATTI~RSSON. E. 13. COVb'LI.MG AND J+ PoR.~.-rl|. Blochz]~|r Rloph.l,s. ,'|eta. 6 7 (1~9b3) 1+ j . GLOMSt-;T Ar¢o J . YORATH, l'l;ochim. Bioph3..*. .r|~ta, 30 ~ltlf',O) J. I J . ~'~ORATH AND ~+ ['~tERTIgN, i n D. Z;L1CH, +~¢]clhc,d_q ,~.f ,ffioche~mi~'al .4nalvs~:i, ~,'ol I X . I n t e r Aclence-Wiley, New Y~rk. London, 1962, p t 9 3 . 7 E . V A ~ S E U R . . - I £ t d l (..'~l~m..~c61nd.. ;I [ t 9 4 ~ ) 6 ~ 3 . • D. H . S]'ACKMAFI, ~V. H+ ,~rl-:l.'~ ~ D S+ .~[ooR rr, ,-),l,t ('.'h~cttt.. 3 o ( ] 9 5 8 ) tic/o+ * S . . X i o o R ~ . i n t h e |)rc.,~'~. to %t,r. j. ARC}IlilALD, J . Ph~s. a n d Cotlo~d. C h e m . 51 !it,_iT) t z o 4 . tt T . L. MCSI1ERKIN &NI.) l i . M.~.R~;I,-IALL..~ci¢~u.t', t ~t~ (1052] [42+ == j . A. G.++/t~.'OIGNI5 A N n M. 51. G.+,iscot(;~,'t':. Biologw.at I)e'~,ra,latic, n o f Celfulo.¢e. ] ~ t t t t c r w o r t h a n d C o . . L o n d o n , t 9 6 o . p. 7 2. ~= 13. A. S T O ~ E . ,\'~+/ure. tI~Z liqh:5) 687+
i¢~twhtm. Bio#hy:i..-Icla. 67 (,,9t~3) 9 t 5
9
BBA X 2 X 2 0
STUDI ES ON CELLULOLYTIC
ENZY MES
II. M U L T I P L I C I T Y O F T H E C E L L t . I L O L Y T I C E N Z Y M E S O F POL Y P O R U S V E R S t C O L O R G,. P E T T E R S S O N
^ND J P O R A T t t
lnstffvCe of 131ochemiMr),, University of I.:pp.~ala, l;ppsulu (Suarden) (R,*,'civt:d M a y z 5 t h . t~li2)
SItMMARY
I. F o u r cellulolytic c o m p o n e n t s h a v e been isolated from c u l t u r e filtrate of t'olylSorus versicolor. z. T h e m o l e c u l a r w e i g h t o f one o f t h e c o m p o n e n t s has been d e t e r m i n e d to be 5 I OflO.
3. A c o m p a r i s o n has bee.n m a d e of t h e a m i n o acid c o m p o s i t i o n s o f the 5x oooc o m p o n e n t a n d t h e p r e v i o u s l y isolated x, o o o - c o m p o n e n t . 4- A n e x p l a n a t i o n for t he o c c u r r e n c e o f m u l t i p l e e n z y m m o f v a r y i n g cellulol}tic a c t i v i t i e s has b ee n p r o p o s e d . lXr,~o,~ucrlox T h e c e l l u l o l y t i c e n z y m e s o f a large n u m b e r of fungi h a v e been purified using block e l e c t r o p h o r e s i s on p o t a t o s t a r c h as d e s c r i b e d b y MASD~I~ a M. t. T h e s e s t u d i e s h a v e s h o w n t h a t m o s t o f t h e fungi s t u d i e d t o daze a p p e a r t o p r o d u c e several cellulolytic c o m p o n e n t s . T h e e x i s t e n c e o f m u l t i p l e c o m p o n e n t s also has been d e m o n s t r a t e d by e l e c t r o p h o r e s i s o f o t h e r e n z y m e s y s t e m s . V a r i o u s e x p l a n a t i o n s h a v e been p r o p o s e d t o a c c o u n t for t h i s h e t e r o g e n e i t y z. F o r e x a m p l e , M I L L E R e t alfl r e c e n t l y h a v e s h o w n t h a t e l e c t r o p h o r e t i c a l l y s e p a r a t e d c o m p , , n e n t s differ in specific a c t i v i t y on c a r b o x y m e t h y l cellulose a n d cel l opent aos e . I t t h e r e f o r e a p p e a r s likely t h a t som e o f t h e cellul o l y tic c o m p o n e n t s m a y p l a y different roles in t h e d e g r a d a t i o n o f cellulose. T h e p r e s e n t w o r k was u n d e r t a k e n to e l u c i d a t e t h e m u l t i p l e - c o m p o n e n t celltdase s y s t e m ot Polyporus versicolor. I t is a cor, t i n u a t i o o o f t h e i n v e s t i g a t i o n r e p o r t e d in t h e p r e c e d i n g a r t i c l e ~. .~tETm)DS
AND IcEst:tA-S
Source of the enzyme T h e m a t e r i a l for t h e p r e s e n t e'~7~'iments wa_~ o b t a i n e d a_~ filtrates f r o m c u l t u r e s Gf t h e w o o d - d e s t r o y i n g b a s i d i o m y c e t e , Polyporus veTs/co/or, gr(,wn in a n t i n e r a l s a l t s m e d i u m w i t h cellulose as sole s our c e o f c a r b o n . T h e fi l t rat e w as c o n c e n t r a t e d a n d f r a c t i o n a t e d on S e p h a d e x as d e s c r i b e d emrlier t, g i v i n g 3 fractions, d e s i g n a t e d B, C a n d D. 13zochim. Hio,phys. Aaa. 67 0 0 ~ , 3 ) .o---J~
IO
G. PETTERSSON, J. PORATH
gnzym~ assays T h e cellulase a c t i v i t y o f all f r a c t i o n s w a s d e t e r m i n e d as t h e a m o u n t o f s o l u b l e reducing substances formed when the enzyme was incubated with cazboxymethyl cellulose or c e l l u l o ~ sr~l. D e t a i l s of t h e a n a l y s i s p r o c e d u r e s were g i v e n p r e v i o u s l y ' .
Zone el~dropkoresis P,rificatio~J of Fyaction B (s~nall scale) Z o n e e l e c t r o p h o r e s i s w a s p e r f o r m e d in a v e r t i c a l b e d s o f P e v i k o n p o w d e r ( P e v i k o n C 870 o b t a i n e d f r o m Fo:~fatbolaget, S t o c k h o l m (Sweden)). T h e m a t e r i a l w a s purified by extensiv-, washing with ethanol, then with 4 M hydrochloric acid and fina!Ly w i t h distilled w a t e r . T h e p o w d e r w a s t h e n s u s p e n d e d in 0.o 5 .~1~ p y r i d i n e a c e t a t e b u f f e r ( p H 4.5). T h e e l e c t r o p h o r e s i s c o l u m n , a z × 35 c m glass t u b e , w a s cln~.d a t t h e b o t t o m w i t h a p l a t e o f p o r o u s p l a s t i c ( V y o n a v a i l a b l e f r o m P o r o u s Plastic Ltd. Dagenham Docks, Essex (England)). The column was provided with a h o r i z o n t a l c a p i l l a r y side t u b e (z m m in d i a m e t e r ) z c m f r o m t h e b o t t o m . T u b i n g o f p o l y x d n y l c h l o r i d e 60 c m in lenh-th a n d z m m inside d i a m e t e r w a s c o n n e c t e d to t h e c a p i l l a r y side t u b e . T h ~ c o l u m n w a s p a c k e d w i t h g l a s s b e a d s ( B a n o t i n i N o . zz) t o a h e i g h t o f z m m a b o v e t h e c a p i l l a r y side a r m . P e v i k o n p a s t e f r e e d f r o m a i r b y e v a c u a t i o n w a s t r a n s f e r r e d to t h e c o l u m n t h r o u g h a p a c k i n g t u b e t o f o r m a 3 o - c m h i g h column. The f~tering properti~ of the bed were tested by passing a narrow zone of dinitrophenylethanolamine through the column.
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Fig. x. Zone eh.ctropherogram of Fraction B. Bed dirw.'nsiona ; × z8 era; buffer o.o5 M pyridineacetate (pH 4.5): currenL 9 mA; voltage. 700 V. Fracttor~q 7-Y4 (1~;) and 3o-36 (Bsl w~re pooled. × - - x , absorbency ~ zSom/~; C ) - ~ , ccllula.se actis'ity against earbo:~ymethyl cetlul~s~ (arbitrary units). Biochi,n.
Biophys÷ At:a,
67 (z963) 9--z 5
CELLULOLYTIC ENZYMES. I!
I!
z o m g o f F r a c t i o n B w e r e d i s s o l v e d in 0. 4 m l 0.05 ,~I p y r i d i n e - a c e t a t c b u f f e r { p H 4-5) a n d p l a c e d o n t o p o f t h e c o l u m n . T h e z o n e w a s a | l o w c d t o f i l t e r d o w n x c m a~xd t h e c o l u m n t h e n a r r a n g e d ;Ls d e s c r i b e d b y POI~ATtt et al. e. E a c h e l e c t r o d e v e s s e l c o n t a i n e d 4 1 o f b u f f e r (0.05 M p y r i d i n e - a c c t a t e ( p H 4-5))- T h e p o l y v i n y l c h l o r i d e t u b i n g w a s c o n n e c t e d w i t h a p e r i s t a l t i c p u m p v i a 0.2 × z o - c m l a t e x t u b i n g . T h e b u f f e r in t h e electro,de v e . ~ e l s w a s m i x e d t w i c e a t s u i t a b l e t i m e i n t e r v a l ~ t o k e e p t h e p H c o n s t a n t . Af':er e l e c t r o p h o r e s i s f o r z o h a t 7o0 V a n d 9 m A . c o n t i n u o u s e l u t i o n w a s b e g u n a t a c o n s t a n t flow r a t e o f 4.5 ml:;h. A f t e r a s e c o n d p e r i o d o f 2o h. electrophoresi.~ w a s s t o p p e d a n d t h e m a t e r i a l on t h e c o l m n n d i s p l a c e d w i t h f r e s h b u f f e r a t a flow r a t e o f x.5 m l / h . F r a c t i o n s c o n t a i n i n g 0.5 m l w e r e c o l l e c t e d . A f t e r d i l u t i o n to z.5 m l t h e a b s o r b a n c y a t z S o nap w a s m e a s u r e d in a B e c k m a n D U s p e c t r o p h o t n m e t e r . T h e r e s u l t s a r e s h o w n in F i g . z a n d r e v e a l t w o a c t i v e f r a c t i o n s : B t a n d I~,~'. T h e d i s t r i b u t i o n o f c d h t l a s c a c t i v i t y a n d a b s o r b a n c y wa~s f [ m n d t o c o i n c i d e o v e r t h e g r e a t e r p a r t o f t h e B~ z o n e . W i t h i n e x p e r i m e n t a l e r r o r , t h e c u r v e s fi~r a c t i v i t y on c e l l u l o s e sol a n d c a r l x ~ x y m e t h y l c e l l u l o ~ ,verc c o i n c i d e n t . T h e f r a c t i o n s w e r e po0Ev3 a s i n d i c a t e d in Fig. t a n d t h e n f r e e z e - d r i e d .
Purification of Fraction B (large sctde) L a r g e r q u a n t i t i e s o f F r a c t i t m B (5t~ m g / m l b u f f e r ) w e r e f r a c t i o n a t e d b y a ~imilar p r o c e d u r e . A ~- × 3 o - c m c o l u m n w a s u s e d . I t wa.~ p r o v i d e d w i t h a c o o l i n g j a c k e t o f methylpolymethacrylate (Perspex) but was not equipped for continuous elution. The e l e e t r o p h o r e s i s t i m e r e q u i r e d a t 400 V a n d 9.2 m A w a s c a l c u l a t e d f r o m t h e r e g u l t s o f e a r l i e r e x p e r i m e n t s . T h u s it w a s p o s s i b l e t o s t o p t h e e l e c t r o p h o r e s i s w h e n t h e f a s t moving component was near the bott,m of the column. After elution, the analysis o f t h e f r a c t i o n s c o l l e c t e d r e v e a l e d t h a t t h e mat~,riM d i s t r i b u t i o n w a s c o m p a r a b l e w i t h t h e e a r l i e r f i n d i n g ~ a s s h o w n in Fig. r.
Purification of Fraction C F r a c t i o n C a ! ~ wa~ f r a c t i t n i a t c d wn t h e ,.amt: c t d u m n a n d in e x a c t l y t h e ~ a m e m a n n e r a s F r a c t i o n B (amMl .~'.xle). T h e p l o t s o f a c t i v i t y a n d a b s o r b a n c y o f t h e v a r i o u s f r a c t i o n s a r e v e r y s i m i l a r t~J tho.-,e fi~r F r a c t i o n B, b u t t h e a c t i v i t y a n d a b sorbancy curves are not coincident. The fraction~ were pooled and freeze-dried as i n d i c a t e d in Fig. 2.
CarbvlLvdrate anal~'sis T h e h e x o ~ c o n t e n t o f c , , m p , m e n t B 1 w a s e s t i m a t e d t o b e z"g.. (w/w) b y t h e o r c i n o l m e t h o d a s d e s c r i b e d b y Vnss~.:uR 7 T h e h e x o s c c o n t e n t o f comp~)ao.nt D p r e v i o u s l y h a d b e e n d e t e r m i n ~ d t o b e 33~',o ( w / w ) I.
Amino acid anMysis " l ~ e a m i n o a c i d c o m p o s i t i o n o f o x i d i z e d .~araples o f c o m I x m e n t ~ Bt a n d D w e r e d e t e r m i n e d u s i n g a B e c k m a n - S p i n c o a u t o m a t i c a m i n o a c i d a n a l y z e r a n d tile p r o c e * T h e e v L z y m c s present in the fractions obt:Lined from g e t f i l l ~ a f i o n or elvctroph~rcsis r e f e r r e d t o u s i a g t h e s a m e labeLq a s t h e f r a c t i o n s ~ t , I l s e t ~ . w b e t h e x t h e y a x e p u x e o r n o t .
will
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F i g . ". Z o n e e l . ~ t r o p h e r o g r a m o f F r a c t i o n C, Bt~l d i m e n ~ o n s , buffex', c u r r e n t a n d v o l t a g e a s i n F i g . t. F r a c t i o n s G - t 4 [C1) a n t i a ~ - 3 1 (Ct) w e r e p o o l e d , X - - × , a b ~ o r b a n c y a t ~ g o m j u ; C>-- - - O , celtulase activity against carboxymethyl cellulose (arbitrary units).
TABI,E I'RFLIMINARY
AItIINO
A minn ~a4~t~
I
A.CID COMPOSITION
Samp~t Bt
OF COMPONKNTS
5a4,ttpt* D
Bit A N D
D
B,~o o/
ami4~ ~£~ (BJD)
I,ymne ! Li~tidine Ammonirt A r girtine C~-~teic acid Aspartic acid .Methionine suifone Threoniue Serine Glutamic acid Proline Glycmc Alaninc VoAine l~ol~ucine LeucLr.e Ty-rozi~e Phen~,[~Lsninc
0-2.57 0.079 o-944 o. [ 5 o o.o3o 0.832 0.o98 o. 4 9 3 o.5z6 o.49o 0.2 to o(,o9 o-4n5 0.265 o. t g 3 o.34I o. x 2 o o.2z 3
0.029 o.ot 5 0-395 o.o3o o.ol 7 o.x605 0.0o8 o. I o 5 x o.zo4z o. t o t o 0.0770 o-o(~¢3 0-0703 o.o744 0.0405 0-0726 Destroyed" 0.0288
* The oxidizing agent was d~,rtroyed using hydrobromi¢
9.0 5.3 z-4 5.o t .8 5.z 12 4- 7 5.0 4.9 2 7 8.9 5-9 3.6 4 +R 4-7 -7-7
a c i d t.
B i o c h i m . B i o p A l t s . ,.iota. 6 7 (196,3) 9 - - t 5
CELLULOLYTIC
ENZY-MI'~S. I I
I3
d u r e o f SPACKblAN d'~ al. s. B e c a u s e o f Lite s m a l l a m o u n t o f m a t e r i M avail;~ble, o n l y a p r e l i m i n a r y d e t e r m i n a t i o n of a m i n o acid c o n t e n t could be made. T h e samples c o n s i s t e d o f f r e e z e - d r i e d m a t e r i a l ~ 2 . 6 o m g o f c o m p o n e n t B , a n d x.2o m g o f c o m p o p,e n t D. T h e h y d r o l y s L g t i m e w a s 24 it. O n e t h i r d o f t h e m a t e r i a J wzLs p l a c e d o n e a c h c o l u m n o f t h e a n a l y z e r . T h e a m i n o a c i d c o m p o s i t i o n s o f t h e .~amplcs are s h o w n in T a b l e I.
Sedime~dation st,dies Sedimentation analysis was performed with a Spinco model E ultracentrifuge p r o v i d e d w i t h a m o d e l D a n a l y t i c a l r o t o r anti a .~ynthetic b o u n d a r y cell. T h e e x p e r i ments were carried out at an average speed of 5')78o rev./min andat a temperature o f 2o °. z m l o f a x~/o (w/v) s o | u t i o n in o.z ,%~ p y r i d i n e a c e t a t e b u f f e r ( p H 5.0) wa~ used. C o m p o n e n t B] g a v e a single s e d i m e n t a t i n g boundary.'. T h e s e d i m e n t a t i , m c o e f f i c i e n t sa0,~o w a s f o u n d t o be 4 . z I $. C o m p ~ m e n t B= w h e n e x a m i n e d u n d e r t h e .,mane c o n d i t i o n s al~o a p p e a r e d t o be homogenecm.~ a n d s~,,o e q u a l t o 4 . z 6 .% w a s o b tairled. 1.:, !: :.f m;-¢,wi;!l .nn,~:!:. ;t T]''C,--'~rcr'." t,~ r,lrl c o r n p r , n e n t C: ;II,'l (:2 iYt ~1 lOW c o n c e n t r a t i o n n a m e l y o . 3 ~ 0 [w,,v). U n d e r the~e ,~ut~,ptimal r o n d i t i o n s it w a s difficult t o d i s c o v e r a n y p o l y d i s p e r s i t y , sz~.~ wa~ f o u n d to be 2.o S for C t a n d r . 9 S h)r C~.
l)etermi~lalion of melecz,lar weight T h e m o l e c u k : r w e i g h t o f c o m i , o n e , t l~ 1 w a s d e t e r m i n e d b y t h e , n e t l , u / o f A R C , I ISAL,,'--". T h e ~.entrifuge cell w;L-~ filled t o a h e i g h t o f xo m m w i t h a t°A) (,.v/x.) .¢,olution tff t h e p r o t e i n .~r. o.1 M p y r i d i n e a c e t a t e ( p H 5-o). T h e a v e r a g e sp~'.ed o f r o t a t i o n w a s Ix 573 r e v . / m i u a t 2o ". T h e m o l e c u l a r w e i g h t e s t i m a t e s in Table. ! I w e r e d e t e r m i n e d "I:~ T]I.E [I M O L E C t T L A R %VF.[GHT Or-" L'~SLLUL.%SE C O M P O N F . N T ~1 F~qrl:.~.XTE[) s~*" 5 5 1 ) I . % l E N T A T I O N .%NAi%'.%IS
The vitriatton among the ~ a : u ~ is l,robabl.v due to u;l
m
]b z4 3-" 4o 48 56
.t,'4 ~.~J
5z 0oo 48 9o.> ..,%.f, Z.~O 5i 3cxl
64 72. 80 ~t~ 9 t'
Average
55 400 53 200
4,'~ 5o0 4 ° Cx.~o 51 2,.,o
51 q o o
.#8 7ix)
51 ~=oo
5 t (x>o
.-5J ~,[x~ 5-~ t ' ~
4 0 .~oo
47
:oo
51 boo
5 2 zo<] 5z ¢)c,,
50 8o0 B~ch*,,. L~iophy¢. Acta. 67
(t963) 9 - ' 5
14
G. P E T T E R S S O N . $, PORATH
from e x t r a p o l a t e d meniscus a n d b o t t o m values, assuming t -- t~ .- o.29. The value o.7x for fi was calculated from the a m i n o acid composition according to MCMEEKIN AND M A R S H A L L i t .
DISCUSS|ON
The rc_~ults of this investigation clearly demon.~trate the occurrence of multiple ccllulolytic enzymes in Polyporjts v~sicolor. T h e e n z y m e c o m p o n e n t s differ in moleculax size a n d differences in substrate specificity h a v e been o b ~ r v e d . In addition to /~-glucosidase, at I~ast 5 cellulolytic c o m p o n e n t s seem to be present in the culture fllt rate. Additional c o m p o n e n t s are indicated b y the p a t t e r n of distribution of a c t i v i t y in the various fractions (Figs. x. 2). We have recognized two pairs o f c o m p o n e n t s moving with slightly different relative velocities on Sephadex G 75 (see ref. 4) a n d with sedimentation coefficients o f 4.2 a n d 2.o S respectively. In a preceding paper 4 it fifth component, designated D, ha-~ been described. L a c k o f material has prevented examination of the p(~,~sible significance of the occurrence of paired enzymes. Only components B l and D have been obtained in quantities sufficient for s e m i q u a n t i t a t i v e a m i n o acid analysis a n d molecular weight determinations. Despite the u n c e r t a i n t y of a m i n o acid analysis, several points of evidence in the results appear w o r t h y of mention. C o m p o n e n t s B t a n d D contained conspicuous a m o u n t s of acidic a m i n o acids. The a m m o n i a c o n t e n t in the h y d r o l y s a t e was very high. Electrophoresis revealed t h a t B t was only slightly positively charged a t p H 4-5 while component D was immobile at this pH, The~se facts t o g e t h e r s t r o n g l y suggest t h a t a large n u m b e r of c a r b o x a m i d e side groups are present in b o t h ce$1ulase components. As sho~vn in Table 1, nine amino acids o u t of sixteen occur in a b o u t the same proportions. These findings suggest t h e existence of c o m m o n polypeptide units. A n u m b e r of cellulolytic organisms produce m u l t i - c o m p o n e n t cellula~e5 tt. I t has been shown t h a t the~e components occasionally differ in specificity on polysaccharides of various molecular sizes. Based on these observations STONE~* propo.~ed the following scheme for the degradation of cellulose. "oliga.~" Cellulose - - ~
cellr~,lextrin ~ - - ~ c e l l o b i u ~
"l~flyasc"
It is evident from the p r e ~ n t work t h a t the basidiomycete Polyporus t'ersicolor produced several cellulolytic components. T h e gel filtration m e t h o d , described in the preceding article 4, gave a fractionation according to molecular size; the low-molecularweight proteins being less mobile t h a n those o f high molecular weibht. These earlier results therefore show, t h a t the ~-glucosidase in F r a c t i o n A, is of higher molecu!~r weight t h a n t h e e n z y m e s pre.sent in Fractions B, C a n d D. T h e l a t t e r e n z y m ~ are active on cellulose and c a x b o x y m e t h y l cellulose. The a c t i v i t y curves for cellulose and c a r b o x y m e t h y l cellulose are displaced in relation to one a n o t h e r in a m a n n e r t h a t suggests t h x t the e n z y m ~ m o s t active on cellulose are o f larger molecular size t h a n those active on carbox.vmethyl cellulose. L a c k of coincidence between these two kinds of a c t i v i t y has been d~rnonstrated evvn more convincingly for cellulolytic enz y m e s of some other organisms (to be published later). These circumstances indicate Bioch~m. B i o p h y s . ,4eta. b 7 ( I 9 6 3 ) 9~-i5
CEI+I.III.fYI+'+'TIC E.~Z~'31E.¢,. II
T~
t h a t an inverse relationship m a y exist b e t w e e n the molecular size o f the e n z y m e c o m p o n e n t s a n d t h a t o f the substrates: L o w - m o l e c u l a r - w e i g h t c e I l u l a ~ s h o w i n g h i g h e s t a c t i v i t y on gluco~idic b o n d s in p o l y s a c c h a r i d e c h a i n s w h i m the h i g h - m o l e c u l a r w e i g h t e n z y m e s , w i t h the /~-gluco~ida,e as t h e mt~t e x t r e m e c~L-;e, preferenti'~lly c a t a l y z e t h e h y d r o l y s i s of oligosaccharictes. %ucL a molecular size r e l a t i o n s h i p s h o u l d facilitate the d e g r a d a t i o n of cellulo.,~ b y ;t geries of e n z y m e s a c t i n g in a se~!uential m a n n e r . To test the v a l i d i t y of this h v p o t h e s i s , t h e ~ i n v e s t i g a t i o n s will be e x t e n d e d t o include cellula_ges from o t h e r fungi, t o g e t h e r w i t h a kinetic characteriz a t i o n o f the c o m l ~ m e n t s i.¢,olated. A C K NO',VI. I-~lit ;E M 16 N'[ S
W e are i n d e b t e d to P r o f e ~ o r g.. "I~sEJAt,~; a n d Dr. E. B. COWLing; fi~r their kind interest. For skilful technica! assistance we wi~h to t h a n k Miss I~. INGEL+MAN. This i n v e s t i g a t i o n wa~ s u p p o r t e d b y a grant from the S w e d i s h N a t u r a l Science l',esear+-I-. Council and from the N a t i o n a l Scien,t. F u u n d a t i o n (G I87O2). REF'ET~ ENCE5 t M+ M ^ I ~ D E L S . G . I.. MlUt.EK a . ~ O Ft. V,'. S t . A r ~ r . J ~ . . :lr¢t,t. t~och,.'m. Riopk.,.'s. 9 3 It0,',1) It F, ~'RODI..X.WSKI, ,-J~tn. P,'.I/, .-lead. Net.. w-! (/,:~,)i! ¢,'.,.-. | G . L . MILLIe.n &Nil It{. BI~,ZG.~.LIS, Al'ch. ~Jiocktt'Jt ~ff*o#ll',.$., 95 (IOG/) | 9 -
It5+
• G, ATTI~RSSON. E. 13. COVb'LI.MG AND J+ PoR.~.-rl|. Blochz]~|r Rloph.l,s. ,'|eta. 6 7 (1~9b3) 1+ j . GLOMSt-;T Ar¢o J . YORATH, l'l;ochim. Bioph3..*. .r|~ta, 30 ~ltlf',O) J. I J . ~'~ORATH AND ~+ ['~tERTIgN, i n D. Z;L1CH, +~¢]clhc,d_q ,~.f ,ffioche~mi~'al .4nalvs~:i, ~,'ol I X . I n t e r Aclence-Wiley, New Y~rk. London, 1962, p t 9 3 . 7 E . V A ~ S E U R . . - I £ t d l (..'~l~m..~c61nd.. ;I [ t 9 4 ~ ) 6 ~ 3 . • D. H . S]'ACKMAFI, ~V. H+ ,~rl-:l.'~ ~ D S+ .~[ooR rr, ,-),l,t ('.'h~cttt.. 3 o ( ] 9 5 8 ) tic/o+ * S . . X i o o R ~ . i n t h e |)rc.,~'~. to %t,r. j. ARC}IlilALD, J . Ph~s. a n d Cotlo~d. C h e m . 51 !it,_iT) t z o 4 . tt T . L. MCSI1ERKIN &NI.) l i . M.~.R~;I,-IALL..~ci¢~u.t', t ~t~ (1052] [42+ == j . A. G.++/t~.'OIGNI5 A N n M. 51. G.+,iscot(;~,'t':. Biologw.at I)e'~,ra,latic, n o f Celfulo.¢e. ] ~ t t t t c r w o r t h a n d C o . . L o n d o n , t 9 6 o . p. 7 2. ~= 13. A. S T O ~ E . ,\'~+/ure. tI~Z liqh:5) 687+
i¢~twhtm. Bio#hy:i..-Icla. 67 (,,9t~3) 9 t 5