Purification of coenzyme a by affinity chromatography

520

Biochimica et Biophysica Acta, 3 3 8 ( 1 9 7 4 ) 5 2 0 - - 5 2 8 © Elsevier S c i e n t i f i c P u b l i s h i n g C o m p a n y , A m s t e r d a m - - P r i n t e d in T h e N e t h e r l a n d s

BBA 27334

PURIFICATION OF COENZYME A BY AFFINITY CHROMATOGRAPHY*

YUHSI MATUO, TETSUYA TOSA and ICHIRO CHIBATA

Department of Biochemistry, Research Laboratory of Applied Biochemistry, Tanabe Seiyaku Co., Ltd, 962 Kashima-cho, Higashiyodogawa-hu, Osaka, (Japan) (Received August 13th, 1973)

Summary 1. The reduced form of coenzyme A (CoA) was immobilized by CNBractivated Sepharose 6B. By chromatography using the immobilized CoA column, it was found that a protein having specific affinity to CoA, CoA-affinity protein, is contained in the dialyzed extracts of the bacteria accumulating CoA. Of the bacteria investigated, Sarcina lutea showed the highest content (approximately 90%) of the protein. 2. The affinity adsorbent, CoA-affinity protein--Sepharose, was prepared by covalently linking the dialyzed extract of Sarcina lutea into Sepharose 6B previously activated with CNBr. Reduced CoA, oxidized CoA, dephospho-CoA, ATP and ADP were adsorbed to the adsorbent column, whereas AMP was not. These compounds adsorbed were eluted by increasing ionic strength (I) with NaC1. When the column was equilibrated with sodium acetate buffer (pH 6.0, •=0.04), reduced CoA was selectively adsorbed. 3. By affinity chromatography employing CoA-affinity protein-Sepharose, reduced CoA of 92% purity was obtained in a yield of 94% from crude reduced CeA of 5% purity. 4. The adsorbing capacity of the adsorbent to reduced CoA was 85 gg per ml of the adsorbent under operating conditions at pH 6--7 and 10--25°C. The capacity of the adsorbent was not changed by storage at 5°C and pH 6.0 for a month.

Introduction Generally, reduced CoA has been purified from microbial cells or fermentation broth by the method of precipitation with heavy metals [1] or by ionic-exchange chromatography [2]. However, these methods are not advanta* Presented at the Annual Meeting of the Kinki Division of the Japanese Biochemical Society, Osaka, Japan, May 2Sth, 1973.

521

geous for mass production of reduced CoA because of their complexity and low yield. On the other hand, recently affinity chromatography has been employed as an advantageous method for the purification of biological substances [3]. In this paper, we describe a new approach for the purification of CoA by the technique of affinity chromatography and a selection method of a ligand in affinity chromatography. Materials and Methods

Materials Sepharose 6B was purchased from Pharmacia Fine Chemicals (Uppsala, Sweden). Reduced CoA was the product of Tanabe Seiyaku Co., Ltd. {Osaka, Japan). Dephospho-CoA and phosphate acetyltranferase (EC 2.3.1.8) were obtained from C.F. Boehringer and Sohne {Mannheim, Germany). ATP, ADP and AMP were the products of Kojin Co., Ltd (Tokyo, Japan). All other chemicals were of analytical grade. Culture o f bacteria Brevibacterium ammoniagenes IAM 1641 was grown with shaking at 30°C for 84 h in the medium (pH 7.0) containing 1% glucose, 1.5% peptone, 0.1% yeast extract, 0.3% K2HPO4, 0.2% NaC1 and 0.02% MgSO4"7H20. Microbacterium flavum IAM 1642 was grown with shaking at 30°C for 84 h in the medium (pH 7.0) containing 5% glucose, 4.4% corn-steep liquor, 1.4% peptone, 0.5% KH2PO4, 0.5% K2HPO4 and 0.1% MgSO4"7H2 O. Micrococcus rubens IFO 3768 was grown with shaking at 30°C for 72 h in the medium (pH 7.0) containing 5% glucose, 1.35% peptone, 1% meat extract, 0.45% yeast extract, 0.55% casamino acid, 0.5% KH2PO4, 0.5% K2 HPO4 and 0.1% MgSO4" 7H2 O. Sarcina lutea IAM 1099 was grown with shaking at 30°C for 72 h in the medium (pH 7.0) containing 5% glucose, 4.4% corn-steep liquor, 1.4% peptone, 1% ammonium acetate, 0.5% KH2PO4, 0.5% K2HPO4 and 0.1% MgSO4" 7H20. Preparation of dialyzed extract The harvested cells were washed twice with 0.9% NaC1 and lyophilized. The lyophilized cells (3 g) were sonicated in suspension in 100 ml of 0.05 M sodium phosphate buffer {pH 7.0) and were centrifuged at 20000 × g for 30 rain. The supernatant was dialyzed against 0.01 M sodium acetate buffer (pH 6.0) for 15 h and used for experiments as the dialyzed extract. Sonication was performed at 20 kcycles (150 W) for I h under cooling by a sonicator of Marine Instruments Co., Ltd, Type T-A-4015 (Tokyo, Japan). Preparation of crude CoA from S. lutea The process for fermentative production of CoA and preparation of crude CoA were perform,~d according to the procedure of Nishimura et al. [4] as follows. After 3 days culture with shaking at 30°C in the same medium (1 1) described above, calcium pantothenate {0.2%), cysteine-HCl {0.2%) and adenine (0.1%) were added to the culture, and the cultivation was continued for 1

522 or 2 days further. The cultured broth was boiled for 8 min and centrifuged. The resulting supernatant was charged onto the charcoal column. After the column was washed with 0.001 M HC1, elution was continued with 40% acetone containing 0.28% ammonia. The column effluent was evaporated under vacuum at 25°C. The resulting concentrated solution (200 ml) was used for experiments as crude CoA.

Preparation of crude reduced CoA To 100 ml of the crude CoA solution described above, 15 ml of 2-mercaptoethanol were added. The mixture was adjusted to pH 8.0 with NaOH and gently stirred for 2 h. After adjusting the pH to 3.0 with HC1, 1.2 1 of ethanol were added to remove 2-mercaptoethanol. The resulting precipitates were collected by centrifugation and dissolved in a small volume of water. Crude reduced CoA solution was prepared by repeating this ethanol treatment twice.

Preparation of oxidized CoA Oxidized CoA was prepared from the above crude CoA solution by chromatography on a DEAE-cellulose column using a linear gradient elution with LiC1 [4]. Oxidized CoA fractions were collected and lyophilized. Lyophilized powder was dissolved in 5 ml of water. To the solution, 50 ml of acetone-methanol (10:1 by vol.) were added. The precipitates produced were collected by centrifugation. The precipitates were dissolved in a small volume of water and charged onto the column of Dowex 50 X8 (H ÷ type). The column effluent was collected and lyophilized. The purity of the oxidized CoA was approximately 95%.

Preparation of CoA--Sepharose column A solution of 3.5 mg of pure reduced CoA in 10 ml of 0.1 M sodium acetate buffer (pH 6.0) was reacted at 25°C for 6 h with 3 g (packed weight) of Sepharose 6B previously activated with CNBr according to the procedure of Ax~n et al. [5]. The mixture was filtered and washed with 10 ml of 1 M NaC1. The a m o u n t of CoA immobilized was estimated by a spectrophotometric determination of the unreacted CoA in the filtrate and washings. The obtained adsorbent contained 3.0 mg of CoA per 3 g (packed wt) of Sepharose 6B. The adsorbent, CoA--Sepharose, was packed into a column (0.6 cm × 9 cm), and used for experiments after washing with 0.1 M sodium acetate buffer (pH 6.0) containing 0.1 M 2-mercaptoethanol.

Preparation of CoA-affinity protein--Sepharose column CNBr-activated Sepharose 6B (1 g packed wt) prepared by the procedure of Ax~n et al. [5] was reacted with 20 ml (20 mg protein) o f the dialyzed extract from S. lutea at pH 8.5. After gentle shaking at 5°C for 6 h, the pH was adjusted to 6.0 by the addition of diluted acetic acid and the mixture was shaken at 5°C for 12 h. To the mixture, another 1 g (packed wt) of CNBractivated Sepharose 6B was added, and then gently shaken at 5°C for 12 h. The mixture was filtered and washed with 20 ml of 1 M NaC1. The a m o u n t of protein immobilized was estimated by determination of the unreacted protein in the filtrate and washings. The obtained adsorbent contained 13.2 mg of protein per 2 g (packed wt) of Sepharose 6B.

523

Determination o f protein. Protein was determined by the m e t h o d of Lowry et al. [ 6 ] , using crystalline bovine albumin as a standard. Determination o f CoA. Reduced CoA was determined spectrophotometrically by the use of phosphate acetyltransferase from Clostridium kluyveri according to the m e t h o d of Michal and Bergmeyer [7], and total CoA (reduced CoA + oxidized CoA) was assayed in the presence of cysteine by the m e t h o d of Stadtman et al. [8] using the same enzyme. The purity of a CoA fraction was determined on the basis of the molecular extinction coefficient at 257 nm [9]. The absorbance was measured at 20--25 ° by a Hitachi--Perkin--Elmer 139 spectrophotometer, using cuvettes of 1-cm optical path. Determination of ionic strength. Ionic strenghts of the buffer and eluate were determined by their conductivity, using an NaC1 solution as a standard. Conductivity was measured at 20- 25°C by a Radiometcr Type CDM 2d conductivity meter and a cell of Type 114 (Copenhagen, Sweden). Results

Chromatography o f dialyzed extracts from bacteria accumulating CoA on a CoA--Sepharose column For the preparation of proteins having an affinity to CoA, four kinds of bacteria accumulating CoA were selected as the protein source. Protein fractions could be prepared from the dialyzed extracts of bacteria accumulating CoA by sonication (Table I). The extraction efficiency of protein was lowest in S. lutea among the bacteria tested. To examine the content of proteins having specific affinity to CoA in the dialyzed extracts from the bacteria, the extracts were chromatographed with a CoA--Sepharose column. After the application of each extract (10 mg protein) to a CoA--Sepharose column, the column was washed with sodium acetate buffer (pH 6.0, I=0.01), and the buffer containing NaC1 of I=0.5 was passed through the column. Table I shows the yields of protein in the fractions adsorbed and n o t adsorbed. When all the fractions not adsorbed were recharged to a second CoA--Sepharose column, no significant adsorption of protein was observed, indicating that the a m o u n t of protein TABLE I A M O U N T OF P R O T E I N AND C O N T E N T OF C o A - A F F I N I T Y P R O T E I N IN D I A L Y Z E D E X T R A C T S FROM BACTERIA ACCUMULATING CoA Dialyzed extract (10 mg protein) was charged onto the CoA--Sepharose c o l u m n previously equilibrated w i t h s o d i u m a c e t a t e b u f f e r ( p H 6.0, I = 0 . 0 1 ) . " N o t a d s o r b e d " i n d i c a t e s the f r a c t i o n e l u t e d w i t h the e q u i l i b r a t i n g b u f f e r , and " A d s o r b e d " i n d i c a t e s the f r a c t i o n e l u t e d w i t h the b u f f e r of I = 0.5. Bacteria accumulating CoA

Sarcina lutea Microbacterium flavum Micrococcus rubens Brevibacteriurn ammoniagenes

A m o u n t of protein in d i a l y z e d e x t r a c t f r o m dried cells ( m g / g o f d r i e d cell)

40 110 250 400

Yield o f p r o t e i n in fractions from CoA-S e p h a r o s e c o l u m n (%) Not adsorbed

Adsorbed

10 35 59 65

88 60 38 28

524 02

P-3

P 4

,

010 z,.

c-

E C

~o.~

<~

u P-1 i

P-2 /~ i

10

20

30

Fraction number Fig. 1. (1 c m eluted buffer

C h r o m a t o g r a p h y o f r e d u c e d C o A o n the C o A - a f f i n i t y p r o t e i n - - S e p h a r o s e c o l u m n . T h e c o l u m n × 5 c m ) was e q u i l i b r a t e d w i t h s o d i u m a c e t a t e b u f f e r ( p H 6.0, I = 0 . 0 1 ) , T h e first p e a k ( P - l ) was w i t h t h e e q u i l i b r a t i n g b u f f e r . Other p e a k s w e r e e l u t e d b y l i n e a r g r a d i e n t e l u t i o n , using 50 m l o f the a n d 50 m l of 0.1 M NaCl. F r a c t i o n v o l u m e was 3 ml. • e, a b s o r b a n c e at 2 6 0 run; ~...~, I.

initially charged was n o t in excess of the adsorption capacity of the adsorbent. A protein fraction adsorbed onto a CoA-Sepharose column is named CoAaffinity protein. It was shown that such protein accounted for approximately 90% in the extract from S. lutea. These results indicate that CoA-affinity protein is effectively obtained from the dialyzed extract of S. lutea without chromatographic separation by a CoA--Sepharose column.

Adsorption of reduced CoA to CoA-affinity protein--Sepharose column Adsorption of reduced CoA to a CoA-affinity protein--Sepharose column was examined. When the column was equilibrated with sodium acetate buffer (pH 6.0, I=0.01), pure reduced CoA was adsorbed and eluted by increasing I with NaC1. As shown in Fig. 1, two minor peaks (P-l, 2) and two major peaks (P-3, 4) were fractionated. By the determination of CoA using phosphate acetyltransferase, it was found that P-1 was an impurity in the CoA sample used, P-2 was oxidized CoA and P-3 and P-4 were reduced CoA. The ionic strength of the fraction showing the maximum content was measured to be 0.025 for P-3 and 0.06 for P-4, respectively.

Adsorption specificity of CoA-affinity protein--Sepharose To clarify the specificity of CoA-affinity protein--Sepharose, oxidized CoA, dephospho-CoA, ATP, ADP and AMP were separately charged onto the column. The ionic strength of the fraction showing the maximum content of these compounds was measured, and the resulting values were superimposed in Fig. 2. All c o m p o u n d s except for AMP were adsorbed onto the adsorbent column of I=0.01. From this result it is expected that when the adsorbent column is equilibrated with the buffer of I - 0 . 0 4 , reduced CoA can be selectively adsorbed.

Purification of reduced CoA by chromatography with CoA-affinity protein-Sepharose column When the crude reduced CoA solution {amount of total CoA: 336 #g) was

525

Not (adsorbt~d

Aclsorbed (elut ed by linear 'gradient method) Red. Co A Red. Co A

\ "~k. \i

Oxid.

.,

co

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0

L

ii;

0.01

0.02

,.

D,r~osp.o-

,tl ;' *"":". i 0.03 0.04 Ionic s t r e n g t h

0.~05

0.06

Fig. 2. A d s o r p t i o n s p e c i f i c i t y of C o A - a f f i n i t y P r o t e i n - S e p h a r o s e . T h e m e t h o d of c h r o m a t o g r a p h y w a s t h e s a m e as in Fig. 1. C h r o m a t o g r a p h i c p a t t e r n s of t h e r e s p e c t i v e c o m p o u n d s w e r e s u p e r i m p o s e d . C o m p o u n d s in e l u a t e s w e r e d e t e r m i n e d s p e c t x o p h o t o m e t r i c a l l y b y m e a s u r i n g t h e a b s o r b a n c e a t 2 6 0 n m . T h e h o r i z o n tal axis in~licates t h e I o f t h e f r a c t i o n s e l u t e d , a n d t h e v e r t i c a l axis i n d i c a t e s t h e a r b i t r a r y u n i t o f absorbance at 260 nm.

applied onto a CoA-affinity protein--Sepharose column equilibrated with the buffer (pH 6 . 0 , / - - 0 . 0 4 ) , the m o s t part of the contaminants with an absorbance at 260 nm were not adsorbed (Fig. 3). The CoA in the fraction not adsorbed was determined as oxidized but not reduced form (Table II). The reduced CoA fraction was eluted from the column by increasing I to 0.1 with NaC1. Summary of the purification of reduced CoA is presented in Table II. The reduced CoA fraction showed 100% purity and 67% yield as total CoA, and 92% purity and 94% yield as reduced CoA.

12 10

E 8 to (.o ¢,4 < 6

4 2 0

50 40 E v

/ 2

30

<

o (J

20

~ o

l0

6 8 Fraction number

10

Fig. 3. A f f i n i t y c h r o m a t o g r a p h y of c r u d e r e d u c e d C o A o n t h e C o A - a f f i n i t y p r o t e i n - - S e p h a r o s e c o l u m n . T h e c o l u m n (1 c m × 5 c m ) w a s e q u i l i b r a t e d w i t h s o d i u m a c e t a t e b u f f e r ( p H 6.0) c o n t a i n i n g NaCI ( I = 0 . 0 4 ) . C r u d e r e d u c e d C o A s o l u t i o n (0.2 m l , 3 3 6 # g as t o t a l C o A ) w a s d i l u t e d to 2 m l w i t h t h e e q u i l i b r a t i n g b u f f e r a n d c h a r g e d o n t o t h e c o l u m n . T h e first p e a k was e l u t e d w i t h t h e e q u i l i b r a t i n g b u f f e r . T h e s e c o n d p e a k was e l u t e d w i t h t h e b u f f e r o f I = 0 . 1 . T h e ionic s t r e n g t h of the b u f f e r w a s c h a n g e d a t t h e a r r o w i n d i c a t e d . F r a c t i o n v o l u m e w a s 3 ml. • "~, a b s o r b a n c e at 2 6 0 n m ; o . . . O , ' c o n t e n t of t o t a l C o A (reduced CoA + oxidized CoA).

526

T A B L E II SUMMARY OF P U R I F I C A T I O N OF CoA C r u d e r e d u c e d C o A s o l u t i o n (0.2 m l ) w a s d i l u t e d w i t h s o d i u m a c e t a t e b u f f e r ( p H 6.0, I = 0 . 0 4 ) , a n d charged onto the CoA-affinity protein--Sepharose column. " N o t a d s o r b e d " indicates the fraction eluted with the e q u i l i b r a t i n g b u f f e r , a n d " A d s o r b e d " i n d i c a t e s t h e f r a c t i o n e l u t e d w i t h t h e b u f f e r o f I = 0.1. T o t a l C o A r e f e r s to o x i d i z e d C o A + r e d u c e d CoA. Fractions

Total C o A

Amount

Crude reduced CoA Not adsorbed Adsorbed

Reduced C o A

(J..J.g)

Purity (%)

Recovery (%)

(J/g)

Amount

Purity (%)

Recovery (%)

336 90 226

8 4 100

100 27 67

221 0 207

5 -92

100 -94

T A B L E III E F F E C T O F p H ON A D S O R B I N G C A P A C I T Y O F R E D U C E D C o A T O C o A - A F F I N I T Y P R O T E I N - SEPHAROSE C o A - a f f i n i t y p r o t e i n - - S e p h a r o s e w a s p a c k e d i n t o the c o l u m n ( b e d v o l u m e : 3 m l ) a n d e q u i l i b r a t e d w i t h s o d i u m a c e t a t e b u f f e r o f p H i n d i c a t e d (I = 0 . 0 4 ) . T o t h e c o l u m n , p u r e r e d u c e d C o A ( 3 0 0 j / g ) dissolved in the equilibrating buffer was charged, and the c o l u m n was washed with the buffer. The adsorbing capacity was s p e c t r o p h o t o m e t r i c a l l y d e t e r m i n e d b y t h e a m o u n t of C o A e l u t e d b y t h e b u f f e r o f I = 0.1. Operating pH

Adsorbing capacity (j/g of r e d u c e d C o A / m l of the adsorbent)

5.0 6.0 '7.0

30 86 84

T A B L E IV E F F E C T OF T E M P E R A T U R E PROTEIN--SEPHAROSE

ON A D S O R B I N G C A P A C I T Y O F R E D U C E D C o A T O C o A - A F F I N I T Y

T h e a d s o r b e n t w a s p a c k e d i n t o a j a c k e t e d c o l u m n ( b e d v o l u m e : 3 ml). T e m p e r a t u r e o f t h e c o l u m n was m a i n t a i n e d b y c i r c u l a t i n g w a t e r a d j u s t e d a t t h e t e m p e r a t u r e i n d i c a t e d . O t h e r c o n d i t i o n s w e r e t h e s a m e as in T a b l e III. Operating temperature (°C)

Adsorbing capacity ( j / g of r e d u c e d C o A / m l of the adsorbent)

10 15 20 25 30 35

82 86 84 82 76 66

527 TABLE V EFFECT OF STORAGE TIME ON THE ADSORBING CAPACITY OF REDUCED CoA TO CoA-AFFINITY PROTEIN--SEPHAROSE T h e a d s o r b e n t w a s s t o r e d i n 0.1 M s o d i u m a c e t a t e b u f f e r ( p H 6 . 0 ) c o n t a i n i n g 1 M NaC1 a t 5 ° C for the i n d i c a t e d p e r i o d . T h e a d s o r b e n t w a s w a s h e d w i t h the b u f f e r o f I = 0 . 0 4 b e f o r e use. O t h e r c o n d i t i o n s w e r e t h e s a m e as in T a b l e III.

Storage time (days)

Adsorbing capacity (]Ag of reduced C o A / m l of the adsorbent)

0 23 33 45 51

89 90 86 76 69

Stability of CoA-affinity protein--Sepharose Effects of operating pH and temperature on adsorbing capacity of reduced CoA with CoA-affinity protein--Sepharose were investigated. As shown in Tables III and IV, the adsorbent was stable under the operating conditions of pH 6--7 and 10--25°C. The adsorbing capacity was about 85 pg/ml of the adsorbent under the conditions of /--0.04, pH 6--7 and 10--25°C. The adsorbent was stable for a month, when stored at 5°C in 0.1 M sodium acetate buffer (pH 6.0) containing 1 M NaC1 (Table V). Discussion For the purification of CoA by the technique of affinity chromatography, selection of the ligand to be immobilized is the most important problem. This problem may be solved by selecting one enzyme requiring CoA as a coenzyme. However, this is not advantageous because of difficulty in selection of the enzyme, and the necessity of purification of the enzyme. On the other hand, it is considered that a ligand in affinity chromatography is not necessarily required to be a purified single substance. Therefore, it is possible to prepare the affinity adsorbent for CoA purification by immobilizing the protein fraction showing specific affinity to CoA. This protein fraction is expected to be obtained by using immobilized CoA. On this standpoint, a new approach for selection of a ligand was performed with immobilized CoA. A s CoA is very labile in alkaline pH, we immobilized reduced CoA with CNBr-activated Sepharose 6B at neutral pH in a short reaction time. The infrared spectrum indicates that CoA is linked to Sepharose 6B in an N-substituted carbamate structure. In order to prepare proteins showing specific affinity to immobilized CoA, the bacteria accumulating CoA were selected as the protein source. Therefore, it was expected that the bacteria accumulating CoA would show a higher content of CoA-affinity protein and lower content of enzyme degradating CoA than the other bacteria. It was found that a special ligand for CoA adsorption, CoA-

528 affinity protein, could be isolated from the dialyzed extracts of various bacteria accumulating CoA by chromatography with the CoA--Sepharose column. Of the bacteria investigated, S. lutea was found to be the most advantageous as the source of CoA-affinity protein, because the dialyzed extract contains CoAaffinity protein in a high c o n t e n t and can be used as a ligand without chromatographic separation by CoA--Sepharose. Therefore, an affinity adsorbent for CoA purification, CoA-affinity protein--Sepharose, was prepared by immobilization of dialyzed extract from S. lutea. Chromatographic behavior of reduced CoA on the CoA-affinity protein--Sepharose column indicated the presence of at least two kinds of CoA-affinity protein in the extract of S. lutea. These two kinds of CoA-affinity protein may be classified as lower affinity protein and higher affinity protein. The result of the adsorption specificity of CoA-affinity protein--Sepharose showed that when lower affinity protein was employed to adsorb reduced CoA, all contaminants in the crude CoA were adsorbed onto the adsorbent column. However, when the adsorbent column was equilibrated with the buffer of I=0.04 so as to use the adsorption capacity of the higher affinity protein in the adsorbent without using the adsorption capacity of the lower affinity protein, reduced CoA could be selectively adsorbed without being contaminated. In fact, reduced CoA of 92% purity was found to be isolated in good yield from the crude sample by affinity chromatography with a CoA-affinity protein--Sepharose column. The biological properties of the two kinds of CoA-affinity protein in the affinity adsorbent are under investigation. Although this paper describes the isolation of CoA, the principle and techniques are evidently applicable to the separation of other biological substances especially in the case of difficulties in the selection and/or preparation of their ligands. Acknowledgements The authors are grateful to Mr T. Takayanagi, Manager of the Development Division for his encouragement in this work. We are also indebted to Miss S. Somekawa for her technical assistance. References 1 2 3 4 5 6 7

Lipmann, ., K a p l a n , N . O . a n d Novelli, G . D . ( 1 9 5 0 ) J. Biol. C h e m . 1 8 6 , 2 3 5 - - 2 4 3 s t a t i t m a n , E . R . a u d Kornberg, A. ( 1 9 5 3 ) J. Biol. C h e m . 2 0 3 , 4 7 - - 5 4 C u a t r e c a s e , P. a n d A n f i n s e n , C.B. ( 1 9 7 1 ) in A n n . Rev. B i o e h e m . 4 0 , 2 5 9 - - 2 7 8 N i s h i m u r a , N., S h i b a t a n i , T., K a k i m o t o , T. a n d C h i b a t a , I., s u b m i t t e d t o Appl. M i c r o b i o l . Ax'en. R., P o r a t h , J. a n d E r n b a c k , S. ( 1 9 6 7 ) N a t u r e 2 1 4 , 1 3 0 2 - - 1 3 0 4 L o w r y , O . H . , R o s e n b r o u g h , N . J . , F a r r , A . L . a n d R a n d a l l , R . J . ( 1 9 5 1 ) J. Biol. C h e m . 1 9 3 , 2 6 5 - - 2 7 5 M i c h a l , G. and B e r g m e y e r , H. ( 1 9 6 3 ) in M e t h o d s o f e n z y m a t i c a n a l y s i s ( B e r g m e y e r , H., e d . ) , pp. 5 1 2 - - 5 2 7 , A c a d e m i c Press, N e w Y o r k 8 S t a d t m a n , E . R . , Novelli, G . D . a n d L i p m a n n , F. ( 1 9 5 1 ) J. Biol. C h e m . 1 9 1 , 3 6 5 - - - 3 7 6 9 B u y s k e , D . A . , H a n d s e h u m a c h e r , R . E . , S c h i l l i n g , E . D . and S t r o n g , F.M. ( 1 9 5 4 ) J. A m . C h e m . Soc. 76, 3575- 3577