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Studies on the chemical modification of staphylococcal enterotoxin B. I. Alkylation and oxidation of methionine residues
279
BIOCHIMICAET BIOPHYSICAACTA BBA 35431 STUDIES ON T H E CHEMICAL MODIFICATION OF STAPHYLOCOCCAL ENTEROTOXIN B I. A L K Y L A T I O N AND O X I D A T I O N OF M E T H I O N I N E R E S I D U E S
FUN SUN CHU AND MERLIN S. BERGDOLL
Food Research Institute and Department of Food Science, University of Wisconsin, Madison, Wisc. 537o6 (U.S.A.) (Received April 28th, 1969)
SUMMARY
The methionine residues in staphylococcal enterotoxin B have been modified with iodoacetic acid and H202. Kinetic analysis of the interaction between iodoacetic acid and enterotoxin B at pH 2.7 reveals that four of the eight residues react at a faster rate. The oxidation of enterotoxin B with H202 was much faster at pH 2.3 than at pH 2. 7. At pH 2.3 seven methionine residues were oxidized. Immunochemical and fluorimetric analyses of the modified enterotoxin B indicate that the conformation of the protein is changed when more than six of the methionine residues are modified by either of these methods. The emetic activity of the toxin was also lost when six methionine residues were modified. Since the modification affects both the biological activity and conformation, the role of methionine residues in staphylococcal enterotoxin B is discussed.
INTRODUCTION
Since the purification of staphylococcal enterotoxin B, a simple basic protein frequently involved in food poisoning outbreaks 1, several new enterotoxins have been isolated and purified 2-4 and some of their physicochemical and biological properties have been determined 5. Because enterotoxin B is easy to purify in relatively large quantities6, 7, it has been studied in much more detail than the others. This enterotoxin contains large amounts of polar amino acids and has two tryptophan residues and one disulfide bond per mole of protein 6,s. In order to understand the role of functional groups in this toxin, selective chemical modifications on the toxin have been carried out in this laboratory as well as in others TM. Six of the 21 tyrosine residues are considered to be "free" and are accessible to the solvent 9. Modification of the free tyrosine with acetylimidazole or tetranitromethane has no effect on the biological activity and conformation of the protein. However, when the abnormal tyrosine residues are Biochim. Biophys. Acta, 194 (1969) 279-286
280
F . S . CHU, M. S. BERGDOLL
modified, the conformation is changed with loss of biological activity. Most of the carboxyl and lysyl groups are evenly distributed on the surface and are relatively accessible to the solvent environment. The charges on these groups appear to play a very significant role in the biological activity as well as the structural conformation of the protein1°, 11. Thus, acetylation and succinylation results in expansion of the molecule and a decrease of the biological activity. Nevertheless, no loss of biological activity was observed when the lysyl residues were guanidinated 10. In addition to one disulfide bond in the protein, enterotoxin B contains 8 moles of methionine. DALIDOWlCZ et al. 12 demonstrated that neither the biological activity nor the conformation had changed when the toxin was reduced and carbaxymethylated. Similar results were observed in this laboratory when the toxin was reduced, carboxymethylated or aminoethylated 13. However, the solubility and biological activity of the toxin was decreased or lost when the toxin was oxidized with performic acid. This led to speculation that the methionine residues in enterotoxin B m a y contribute in some way to the biological activity. In the present study the methionine residues in the protein were selectively modified with iodoacetic acid and with H202. The effect of alkylation and oxidation on the conformation and biological activity is presented. MATERIALS AND METHODS
Materials
Enterotoxin B and enterotoxin B antisera were prepared as described previously 5. Reagent grade H202 was obtained from Allied Chemical Co. Iodoacetic acid was obtained from Aldrich Chemical Co. (Milwaukee, Wisc.) and was further recrystallized from hexane before using. The CM-cellulose was Selectacel CM-cellulose No. 77, Type 20 (Schleicher and Schuell Co.) and was prepared according to PETERSON AND SOBER14. Phenol reagent was obtained from Fisher Scientific Co. A nalyses
The amino acid composition was determined on a Spinco Beckman model 12o B amino acid analyzer as previously described 6. The amount of methionine residues modified b y either oxidation or alkylation were determined b y the procedures of NEUMANN et al. 15. In the first case, the oxidized protein was extensively alkylated at pH 2.o, then dialyzed, lyophilized, and oxidized with performic acid which was followed by hydrolysis in 5.7 M redistilled HC1. Oxidation of the S-carboxymethylated enterotoxin B and the control samples was done with performic acid according to the procedure of HIRS16. The quantitative precipitin test and monkey feeding experiments were performed according to the method described previously1, 9. The residual activity was calculated from the total protein precipitated at the equivalent concentration of the modified enterotoxin B divided by the total protein precipitated at the equivalent concentration of the native enterotoxin B (ref. 9)Fluorescence intensity of the native and modified enterotoxin B was determined in an Aminco-Bowman spectrofluorimeter, equipped with a thermostated cell holder which was maintained at 25 °, and except where otherwise stated, the wavelengths of excitation and emission were 285 m/~ and 335 m#, respectively. All the measurements were made with enterotoxin B solutions at the concentration near 3" IO-~ M in o.16 M Biochim. Biophys. Acta, 194 (1969) 279-286
MODIFICATION OF METHIONINE RESIDUES IN ENTEROTOXlN B
281
KC1 and 0.05 M sodium phosphate buffer (pH 6.0). For comparison purposes, the fluorescence readings were converted to an enterotoxin B concentration with an absorbance of I.OO at 277 m# at an arbitrary meter setting (multiplier value of I.OO).
Alkylation with iodoacetic acid Alkylation of enterotoxin B with iodoacetic acid was carried out according to NEUMANN et al. 15. 50 mg of enterotoxin B in 6 ml of distilled water was incubated with 5 ° mg of recrystallized iodoacetic acid at pH 2.7 and 37 °. I-ml aliquots were taken from the incubation mixture at zero time, 30 min, I, 2.5 and 5 h. Each sample was immediately transferred to a CM-cellulose column (0. 9 cm × 30 cm) equilibrated with o.oi M sodium phosphate buffer (pH 5.7). The columns were washed with the equilibrating buffer to zero absorbance at 277 m/~, (pH 5.7) and a constant resistance. Stepwise elution with 0.05 M sodium phosphate buffer (pH 6.8o) and 0.2 M Na2HPO * was then performed. The modified enterotoxin B was eluted from the column with the 0.2 M Na, H P O 4. The samples were dialyzed against 41 of water for 48 h with 4 changes of water and lyophilized. Oxidation with H20 ~ In a typical experiment, 42 mg enterotoxin B in 6 ml of o.3 M H20 ,, pH 2.3o, was incubated at 37 ° for 12 h. An aliquot of i ml was taken from the incubation mixture at certain intervals, diluted with 4 ml of cold distilled water, adjusted to p H 6-7 and lyophilized immediately. Each of the samples was then dissolved (or suspended) in 5 ml distilled water and relyophilized. This procedure was repeated 2 times. A control sample was carried out under the same conditions except in the absence of H,O,. In a separate experiment the solution was adjusted to p H 2.7 ° instead of pH 2.30 for oxidation. RESULTS
Alkylation of enterotoxin B with iodoacetic acid After alkylation at p H 2.70, the modified enterotoxin B was more strongly absorbed b y the CM-cellulose, therefore, the unmodified and partially modified toxin could be readily separated. Fig. I shows the samples which were reacted with iodoacetic acid for different lengths of time. The unreacted iodoacetic acid was washed out with the initial buffer, whereas the unreacted or partially modified enterotoxin B (less than 2 moles per mole) was eluted from the column with 0.05 M sodium phosphate buffer (pH 6.85). Amino acid analysis indicated that the material obtained from the second peak was predominately S-carboxymethyl methionium derivative of enterotoxin B. The amino acid composition of a 5-h alkylated product obtained from the second peak of the CM-cellulose column chromatography, is given in Table I. All eight of the methionine residues were modified. No methionine sulfone was detected in this product when the sample was alkylated and then oxidized. A large number of tyrosine residues was apparently destroyed during the performic acid oxidation. The kinetics of alkylation of methionine residues in enterotoxin B was analyzed according to the method described by RAY AND KOSHLAND17,18. Fig. 2A shows the rate of methionine residues modified and Fig. 2B indicates the rate of decrease of antigen-antibody reaction. Since the reaction was found to be pseudo first order Biochim. Biophys. Acta, 194 (1969) 279-286
282
F.S.
CHU, M. S. B E R G D O L L
TABLE I AMINO ACID
COMPOSITION
OF MODIFIED
ENTEROTOXIN
g
A m i n o a c i d r e s i d u e s p e r mole o f e n t e r o t o x i n B a f t e r d i f f e r e n t t r e a t m e n t s . T h e c a l c u l a t i o n s w e r e b a s e d o n 22 moles o f g l u t a m i c a c i d , 9 moles o f g l y c i n e , 5 moles o f a l a n i n e , 7 m o l e s o f p r o l i n e , 17 m o l e s o f v a l i n e , 9 moles o f isoleucine, a n d 18 moles o f leucine, p e r m o l e o f e n t e r o t o x i n B.
A m i n o acid
No treatment*
A lkylation
A lkylation and performic acid oxidation
HzO ~ and extensive alhylation and performic acid oxidation
Lysine Histidine Ammonia Homoserine lactone Arginine Cysteic acid Methionine sulfoxides Aspartic acid Methionine sulfone Threonine Serine Homoserine Glutamic acid Proline S-Carboxymethyl homocysteine Glycine Alanine Half cystine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Methionine and related residues
33 5 29 o 5 o o 47 o 13 14 o
33 5 33 1.74 5 o o 47 0.64 13 14 --
33 5 4.80 5 1.84 o 46.6 o 13. I 14 --
33 4.53 43.16 1.27 5.13 1.92 o 46-85 6.59 12.66 14 -
22
22
21.92
22
7 o io 5 2 17 8 9 18 21 13 8
7 2.38 lO.85 5.77 trace 17 i .9 ° 9.93 17.oo 21 13 6.66
6.85 1.94 i i. i o 6.2o o 16. oo i .29 9.67 17.76 6.15 12.9 ° 8.03
6.76 o i 1.73 6.32 o 16.97 0.097 9. lO 17.45 12.63 12.6 i 7-95
* O b t a i n e d f r o m BERGDOLL et al. ~ a n d u n p u b l i s h e d o b s e r v a t i o n s o f H u a n g a n d B e r g d o l l of this l a b o r a t o r y .
w i t h respect to m e t h i o n i n e groups, the n o n l i n e a r i t y of the plot indicates t h a t n o t all the m e t h i o n i n e groups are e q u a l l y reactive. I t was a s s u m e d t h a t t h e r e are two classes of m e t h i o n i n e groups, i.e., " f a s t " a n d " s l o w " reacting in the enterotoxin. A b o u t 4 m e t h i o n i n e residues (50%) were a l k y l a t e d at a faster tate. The r a t e c o n s t a n t s for the fast reaction of modification a n d i n a c t i v a t i o n are 0.022 rain -1 a n d 0.043 m i n - L respectively. The "slow" r a t e c o n s t a n t s of modification a n d i n a c t i v a t i o n are 0.004 rain -1 a n d 0.003 rain -1, respectively. The precipitin reaction was diminished to less t h a n 15 % when all eight of the m e t h i o n i n e residues were modified.
Oxidation with H~O~ The reaction of h y d r o g e n peroxide with e n t e r o t o x i n B is shown in Fig. 3. T h e slow and fast r a t e c o n s t a n t s for o x i d a t i o n of m e t h i o n i n e in e n t e r o t o x i n B at p H 2.3 are 0.0023 min -1 a n d o.o21 min 1, respectively. A p p r o x i m a t e l y 6 m e t h i o n i n e residues are oxidized at a faster rate. T h e amino acid composition of an oxidized e n t e r o t o x i n B indicates t h a t n o t all the methionine residues are oxidized even after 12 h of reaction. Biochim. Biophys. Acta, 194 (1969) 2 7 9 - 2 8 6
283
MODIFICATION OF METHIONINE RESIDUES IN ENTEROTOXIN B 100
80 60 ~ e t
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st' Met groups
J
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I 30 T U B E NUMBER
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56 TIME
I 2 3 (HOURS)
5
6
Fig. I. C h r o m a t o g r a p h y of a l k y l a t e d e n t e r o t o x i n B on CM-cellulose. T h e elution p a t t e r n s were o b t a i n e d f r o m s a m p l e s w h i c h h a v e b e e n r e a c t e d w i t h iodoacetic acid for zero h o u r (top), 2.5 h (middle) a n d 5 h (bottom). S a m p l e after r e a c t i o n was directly t r a n s f e r r e d into a 0.9 c m X 3 ° c m CM-cellulose c o l u m n , e q u i l i b r a t e d w i t h o.oi M s o d i u m p h o s p h a t e buffer (pH 5.7). A f t e r w a s h i n g w i t h o.oi M s o d i u m p h o s p h a t e buffer (pH 5-7), to zero a b s o r b a n c e a t 277 m/z, stepwise elution w i t h o.05 M s o d i u m p h o s p h a t e buffer (pH 6.8) (Gt) a n d N a , H P O ~ (G,) (started a t arrow) was p e r f o r m e d . Flow rate, 5.4 m l / t u b e per 2 min. Fig. 2. S e m i l o g a r i t h m i c plot for t h e r e a c t i o n of iodoacetic acid w i t h e n t e r o t o x i n ]3. (A) Loss of m e t h i o n i n e d u r i n g modification. (B) Loss of a n t i g e n - a n t i b o d y r e a c t i o n d u r i n g t h e modification. T h e excess r e a g e n t s were r e m o v e d b y c h r o m a t o g r a p h y a n d dialysis. T w o classes of m e t h i o n i n e g r o u p s were a s s u m e d .
I00 80:
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Total Met groups
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Fig. 3. S e m i l o g a r i t h m i c plot for t h e r e a c t i o n of H~O 2 w i t h e n t e r o t o x i n B. (A) R e a c t i o n w h i c h w a s carried o u t a t p H 2. 3. (B) Loss of a n t i g e n - a n t i b o d y r e a c t i o n w h e n t h e r e a c t i o n was carried o u t a t p H 2.70. Fig. 4. Precipitin a n a l y s e s of e n t e r o t o x i n B a n d modified e n t e r o t o x i n . C u r v e A is a control sample. C u r v e s B - D s h o w a l k y l a t e d e n t e r o t o x i n 13 in w h i c h 2 (Curve B), 6 (Curve C), a n d 8 (Curve D) m e t h i o n i n e residues h a v e been modified, respectively.
Biochim. Biophys. Acta, 194 (1969) 279-286
284
F.s.
CHU, M. S. BERGDOLL
T A B L E II EFFECT
OF CHEMICAL
STAPHYLOCOCCAL
MODIFICATION
ENTEROTOKIN
OF THE
METHIONINE
RESIDUES
ON T H E
EMETIC
ACTIVITY
OF
B
Number of methionine residues modified
Methods of modification
Enterotoxin B (#g /monkey)
Activity*
None 6 8
None O x i d a t i o n b y H=O 2 A l k y l a t i o n b y iodoacetic acid
20 5° 5°
4-6]6 o/6 0/6
* N u m b e r of m o n k e y s s h o w i n g positive r e a c t i o n vs. n u m b e r of m o n k e y s challenged.
T a b l e I shows t h a t o n l y 7 m e t h i o n i n e residues were oxidized after t h e 12 h of reaction. A f t e r I h of reaction aggregation was o b s e r v e d when the solutions were neutralized. The rate c o n s t a n t for the i n a c t i v a t i o n of a n t i g e n - a n t i b o d y reaction is 0.020 min -1 when the reaction was carried out at p H 2.30. However, t h e r a t e c o n s t a n t s are 0.0006 min -1 a n d O.Ol 4 min -1 for t h e slow a n d fast i n a c t i v a t i o n when the r e a c t i o n was carried o u t at p H 2.7. Since the i n a c t i v a t i o n is m u c h slower at p H 2. 7 t h a n at p H 2.3 (rate c o n s t a n t s are o.ooo6 min -1 a n d O.Ol 4 min -1 for the slow a n d fast i n a c t i v a t i o n , respectively), t h e amino acid composition of the p r o d u c t o b t a i n e d at p H 2.7 has n o t been analyzed. 2.0
i
i
A
IOO
hJ ~1 .5
9O 80
70
iG°
LL 1.0
=_>
_j 5o
d
4o
B 30
0.5
# 2o 1.0 I
250
270
290
310
33,0
WAVELENGTH
350
1570 2,90
410
430
2
3
4
5
6
7
8
NO. OF METHIONINE RESIDUES MODIFIED PER MOLE OF ENTEROTOXIN B
(my.)
Fig. 5. Fluorescence s p e c t r a of e n t e r o t o x i n B a n d modified e n t e r o t o x i n B. C u r v e A r e p r e s e n t s t h e control sample. Curves B--D are a l k y l a t e d e n t e r o t o x i n B in w h i c h 2,5 a n d 8 residues of m e t h i o nine were modified. C u r v e E (dotted line) r e p r e s e n t s a n oxidized s a m p l e in w h i c h 5 residues of m e t h i o n i n e were oxidized b y H=O=. Fig. 6. Effect of chemical modification of m e t h i o n i n e residues on t h e a n t i g e n - a n t i b o d y reaction, a n d fluorescence i n t e n s i t y (335 m/~) of enterotoxin. [] a n d Ill, a n t i g e n - a n t i b o d y r e a c t i o n a n d fluorescence i n t e n s i t y of o x i d a t i o n p r o d u c t s , respectively. C) a n d 0 , a n t i g e n - a n t i b o d y reaction a n d fluorescence i n t e n s i t y of t h e a l k y l a t e d p r o d u c t s , respectively.
Biochim. Biophys. Acta, 194 (1 ~69) 279-286
MODIFICATION OF METHIONINE RESIDUES IN ENTEROTOXIN B
285
Effect of modification of methionine residues on the biological activity of enterotoxin B A typical precipitin curve of the modified and unmodified enterotoxin B is shown in Fig. 4. Modification of the methionine residues by either alkylation or oxidation resulted in a significant decrease of antigen-antibody reaction when more than 5 or 6 residues of methionine are chemically modified. The results of the monkey feeding tests are summarized in Table II. The emetic activity was lost when 6 or more residues of methionine were modified. Fig. 5 shows the fluorescence spectrum of the modified and unmodified enterotoxin B. The relationship between the decrease of antigen-antibody reaction and the quenching of fluorescence after oxidation or alkylation is presented in Fig. 6. Neither the antigen-antibody reaction nor t r y p t o p h a n fluorescence was decreased when a control sample was held at pH 2. 3 for 12 h. DISCUSSION
The experimental results indicate that methionine residues in enterotoxin B play a very important biochemical role. Amino acid composition analysis indicated that only methionine residues were altered after alkylation. The apparent loss of tyrosine residues was due to destruction by performic acid during analysis15, in because no loss of tyrosine residues was observed when the alkylated sample was hydrolyzed without performic acid oxidation. The amino acid analysis also suggested that the oxidation of methionine sulfur is the sole chemical modification of significancet hat occurred during the oxidation with H202 (last column of Table I). Although the disulfide bond might have been oxidized during the reaction, evidence has been obtained 1~,13 which indicates the disulfide bond in enterotoxin B is not essential for its biological activity. Kinetic analyses reveal at least 2 or 3 types of methionine residues in enterotoxin B. At approximately p H 2.7, 4 residues are probably located on the surface of the toxin. These 4 residues reacted with iodoacetic acid much faster at p H 2. 7 than the othersXV,TM and were almost instantly oxidized by the H,O, at p H 2. 3. Enterotoxin B m a y undergo a minor conformational change at pH 2. 3 which results in a faster reaction with HzO 2. No significant changes in the fluorescence properties and antigen-antibody reaction have been observed when 4 residues of methionine were oxidized. However, significant changes were observed when they were alkylated. This difference might be due to the alteration of the charges by the alkylation since the chromatographic behavior of the alkylated products has been significantly altered. Major conformational changes (loss of fluorescence) and loss of biological activity occurred when the 5th to 7th methionine residues were modified. These residues m a y play a predominant role in the structural and biological function of the enterotoxin. One methionine residue probably was buried inside of the molecule. Correlation of loss or decrease of antigen-antibody reaction with the change of conformation has been demonstrated in m a n y other proteins such as pepsin and pepsinogen 19,2° and hemoglobin and myoglobin 21 systems. The quenching of fluorescence in proteins after denaturation has been considered also to be a result of the conformational changes 2.. BOROFF AND FITZGERALD ~3 demonstrated the correlation between the loss of biological activity and the loss of tryptophan fluorescence of botulinum toxin. The correlation between the loss of antigen-antibody precipitin reaction and the quenching of fluorescence in the present study can be interpreted as a significant change of conformation. Since present knowledge about the mode of Biochim. Biophys. Acta, 194 (1969) 279-286
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F . s . CHU, M. S. BERGDOLL
action of enterotoxin B and the conformation of the toxin in vitro remains unclear, whether the modified methionine residues are those on the active site is uncertain. However, from the observation of the complete restoration of the activity of the control samples which have been incubated at pH 2.3 for 12 h, and from the pH of the enterotoxin B receptor site which normally is slightly basic, it is clear that the inactivation is due to the conformational change rather than the effect on the active site. Enterotoxin B probably failed to fold to the original conformation after modification and thus lost its emetic activity. The conformational change may either render the susceptibility of enterotoxin B to enzymic digestion or alter the enterotoxin structure to a configuration which fails to interact with the target tissue. ACKNOWLEDGMENTS
The authors wish to acknowledge the technical assistance of Elizabeth Crary. This investigation was supported by public health service grant No. AI o7615 from the National Institute of Allergy and Infectious Diseases. REFERENCES I 2 3 4 5 6 7 8 9 IO ii 12 13 14 15 16 17 18 19 20 21 22 23
S. BERGDOLL, H. SUGIYAMA AND G. M. DACK, Arch. Biochem. Biophys., 85 (1959) 62. S. CHO, K. THADHANI, E. J. SCHANTZ AND M. S. BERGDOLL, Biochemistry, 5 (1966) 3281. R. BORJA AND M. S. BERGDOLL, Biochemistry, 6 (1967) 1467. M. AVENA AND M. S. BERGDOLL, Biochemistry, 6 (1967) 1474. S. BERGDOLL, in R. I. MATELES AND G. N. WOGAN, Biochemistry of Some Foodborne Microbial Toxins, M.I.T. Press, Cambridge, Mass., 1967, p. I. M. S. BERGDOLL, F. S. CHU, I.-Y. HUANG, C. ROWE AND T. SHIH, Arch. Biochem. Biophys., 112 (1965) lO4. E. J. SC~ANTZ, W. G. ROESSLER, J. WAGMAN, L. SPERO, D. A. DUNNERY AND M. S. BERGDOLL, Biochemistry, 4 (1965) IOli. L. SPERO, D. STEFANYE, P. I. BRECHER, H. M. JACOB¥, J. E. DALIDOWICZ AND E. J. SCIIANTZ. Biochemistry, 4 (1965) lO24. F. S. CHU, J. Biol. Chem., 243 (I968) 4342 . F. S. CHU, E. CRARY AND M. S. BERGDOLL, Biochemistry, 8 (1969) 2890. F. S. CHU AND E. CRARY, Biochim. Biophys. Acta, 194 (1969) 287. J. E. DALIDOWICZ, S. J. SILVERMAN, E. J. SCHANTZ, D. STEFANYE AND L. SPERO, Biochemistry, 5 (1966) 2375. I.-Y. HUANG AND M. S. BERGDOLL, Arch. Biochem. Biophys., 132 (1959) 423 . E. A. PETERSON AND H. A. SOBER, J. Am. Chem. Soe., 78 (1956 ) 751 . N. P. NEUMANN, S. MOORE AND W. I-I. STEIN, Biochemistry, I (1962) 68. C. H. W. HIRS, J. Biol. Chem., 2I 9 (1956) 611. W. J. RAY, JR. AND D. E. KOSHLAND, JR., Broohhaven Syrup. Biol., 13 (196o) 135. W. J. RAY, JR. AND D. E. KOSHLAND, JR., J. Biol. Chem., 236 (1961) 1973. H. VAN VUNAKIS, H. LEHRER, W. S. ALLISON AND L. LEVlNE, J. Gen. Physiol., 46 (1963) 589. J. F. GERSTEIN, H. VAN VUNAKIS AND L. LEVlNE, Biochemistry, 2 (1963) 964. M. REICHLIN, N[. HAY AND L. LEVlNE, Biochemistry, 2 (1963) 971. R. F. STEINER AND H. EDELHOCH, Nature, 192 (1961) 873. D. A. BOROFF AND J. E. FITZGERALD, Nature, 181 (1958) 751. M. F. C. R. M.
Biochim. Biophys. Acta, 194 (1969) 279-286
BIOCHIMICAET BIOPHYSICAACTA BBA 35431 STUDIES ON T H E CHEMICAL MODIFICATION OF STAPHYLOCOCCAL ENTEROTOXIN B I. A L K Y L A T I O N AND O X I D A T I O N OF M E T H I O N I N E R E S I D U E S
FUN SUN CHU AND MERLIN S. BERGDOLL
Food Research Institute and Department of Food Science, University of Wisconsin, Madison, Wisc. 537o6 (U.S.A.) (Received April 28th, 1969)
SUMMARY
The methionine residues in staphylococcal enterotoxin B have been modified with iodoacetic acid and H202. Kinetic analysis of the interaction between iodoacetic acid and enterotoxin B at pH 2.7 reveals that four of the eight residues react at a faster rate. The oxidation of enterotoxin B with H202 was much faster at pH 2.3 than at pH 2. 7. At pH 2.3 seven methionine residues were oxidized. Immunochemical and fluorimetric analyses of the modified enterotoxin B indicate that the conformation of the protein is changed when more than six of the methionine residues are modified by either of these methods. The emetic activity of the toxin was also lost when six methionine residues were modified. Since the modification affects both the biological activity and conformation, the role of methionine residues in staphylococcal enterotoxin B is discussed.
INTRODUCTION
Since the purification of staphylococcal enterotoxin B, a simple basic protein frequently involved in food poisoning outbreaks 1, several new enterotoxins have been isolated and purified 2-4 and some of their physicochemical and biological properties have been determined 5. Because enterotoxin B is easy to purify in relatively large quantities6, 7, it has been studied in much more detail than the others. This enterotoxin contains large amounts of polar amino acids and has two tryptophan residues and one disulfide bond per mole of protein 6,s. In order to understand the role of functional groups in this toxin, selective chemical modifications on the toxin have been carried out in this laboratory as well as in others TM. Six of the 21 tyrosine residues are considered to be "free" and are accessible to the solvent 9. Modification of the free tyrosine with acetylimidazole or tetranitromethane has no effect on the biological activity and conformation of the protein. However, when the abnormal tyrosine residues are Biochim. Biophys. Acta, 194 (1969) 279-286
280
F . S . CHU, M. S. BERGDOLL
modified, the conformation is changed with loss of biological activity. Most of the carboxyl and lysyl groups are evenly distributed on the surface and are relatively accessible to the solvent environment. The charges on these groups appear to play a very significant role in the biological activity as well as the structural conformation of the protein1°, 11. Thus, acetylation and succinylation results in expansion of the molecule and a decrease of the biological activity. Nevertheless, no loss of biological activity was observed when the lysyl residues were guanidinated 10. In addition to one disulfide bond in the protein, enterotoxin B contains 8 moles of methionine. DALIDOWlCZ et al. 12 demonstrated that neither the biological activity nor the conformation had changed when the toxin was reduced and carbaxymethylated. Similar results were observed in this laboratory when the toxin was reduced, carboxymethylated or aminoethylated 13. However, the solubility and biological activity of the toxin was decreased or lost when the toxin was oxidized with performic acid. This led to speculation that the methionine residues in enterotoxin B m a y contribute in some way to the biological activity. In the present study the methionine residues in the protein were selectively modified with iodoacetic acid and with H202. The effect of alkylation and oxidation on the conformation and biological activity is presented. MATERIALS AND METHODS
Materials
Enterotoxin B and enterotoxin B antisera were prepared as described previously 5. Reagent grade H202 was obtained from Allied Chemical Co. Iodoacetic acid was obtained from Aldrich Chemical Co. (Milwaukee, Wisc.) and was further recrystallized from hexane before using. The CM-cellulose was Selectacel CM-cellulose No. 77, Type 20 (Schleicher and Schuell Co.) and was prepared according to PETERSON AND SOBER14. Phenol reagent was obtained from Fisher Scientific Co. A nalyses
The amino acid composition was determined on a Spinco Beckman model 12o B amino acid analyzer as previously described 6. The amount of methionine residues modified b y either oxidation or alkylation were determined b y the procedures of NEUMANN et al. 15. In the first case, the oxidized protein was extensively alkylated at pH 2.o, then dialyzed, lyophilized, and oxidized with performic acid which was followed by hydrolysis in 5.7 M redistilled HC1. Oxidation of the S-carboxymethylated enterotoxin B and the control samples was done with performic acid according to the procedure of HIRS16. The quantitative precipitin test and monkey feeding experiments were performed according to the method described previously1, 9. The residual activity was calculated from the total protein precipitated at the equivalent concentration of the modified enterotoxin B divided by the total protein precipitated at the equivalent concentration of the native enterotoxin B (ref. 9)Fluorescence intensity of the native and modified enterotoxin B was determined in an Aminco-Bowman spectrofluorimeter, equipped with a thermostated cell holder which was maintained at 25 °, and except where otherwise stated, the wavelengths of excitation and emission were 285 m/~ and 335 m#, respectively. All the measurements were made with enterotoxin B solutions at the concentration near 3" IO-~ M in o.16 M Biochim. Biophys. Acta, 194 (1969) 279-286
MODIFICATION OF METHIONINE RESIDUES IN ENTEROTOXlN B
281
KC1 and 0.05 M sodium phosphate buffer (pH 6.0). For comparison purposes, the fluorescence readings were converted to an enterotoxin B concentration with an absorbance of I.OO at 277 m# at an arbitrary meter setting (multiplier value of I.OO).
Alkylation with iodoacetic acid Alkylation of enterotoxin B with iodoacetic acid was carried out according to NEUMANN et al. 15. 50 mg of enterotoxin B in 6 ml of distilled water was incubated with 5 ° mg of recrystallized iodoacetic acid at pH 2.7 and 37 °. I-ml aliquots were taken from the incubation mixture at zero time, 30 min, I, 2.5 and 5 h. Each sample was immediately transferred to a CM-cellulose column (0. 9 cm × 30 cm) equilibrated with o.oi M sodium phosphate buffer (pH 5.7). The columns were washed with the equilibrating buffer to zero absorbance at 277 m/~, (pH 5.7) and a constant resistance. Stepwise elution with 0.05 M sodium phosphate buffer (pH 6.8o) and 0.2 M Na2HPO * was then performed. The modified enterotoxin B was eluted from the column with the 0.2 M Na, H P O 4. The samples were dialyzed against 41 of water for 48 h with 4 changes of water and lyophilized. Oxidation with H20 ~ In a typical experiment, 42 mg enterotoxin B in 6 ml of o.3 M H20 ,, pH 2.3o, was incubated at 37 ° for 12 h. An aliquot of i ml was taken from the incubation mixture at certain intervals, diluted with 4 ml of cold distilled water, adjusted to p H 6-7 and lyophilized immediately. Each of the samples was then dissolved (or suspended) in 5 ml distilled water and relyophilized. This procedure was repeated 2 times. A control sample was carried out under the same conditions except in the absence of H,O,. In a separate experiment the solution was adjusted to p H 2.7 ° instead of pH 2.30 for oxidation. RESULTS
Alkylation of enterotoxin B with iodoacetic acid After alkylation at p H 2.70, the modified enterotoxin B was more strongly absorbed b y the CM-cellulose, therefore, the unmodified and partially modified toxin could be readily separated. Fig. I shows the samples which were reacted with iodoacetic acid for different lengths of time. The unreacted iodoacetic acid was washed out with the initial buffer, whereas the unreacted or partially modified enterotoxin B (less than 2 moles per mole) was eluted from the column with 0.05 M sodium phosphate buffer (pH 6.85). Amino acid analysis indicated that the material obtained from the second peak was predominately S-carboxymethyl methionium derivative of enterotoxin B. The amino acid composition of a 5-h alkylated product obtained from the second peak of the CM-cellulose column chromatography, is given in Table I. All eight of the methionine residues were modified. No methionine sulfone was detected in this product when the sample was alkylated and then oxidized. A large number of tyrosine residues was apparently destroyed during the performic acid oxidation. The kinetics of alkylation of methionine residues in enterotoxin B was analyzed according to the method described by RAY AND KOSHLAND17,18. Fig. 2A shows the rate of methionine residues modified and Fig. 2B indicates the rate of decrease of antigen-antibody reaction. Since the reaction was found to be pseudo first order Biochim. Biophys. Acta, 194 (1969) 279-286
282
F.S.
CHU, M. S. B E R G D O L L
TABLE I AMINO ACID
COMPOSITION
OF MODIFIED
ENTEROTOXIN
g
A m i n o a c i d r e s i d u e s p e r mole o f e n t e r o t o x i n B a f t e r d i f f e r e n t t r e a t m e n t s . T h e c a l c u l a t i o n s w e r e b a s e d o n 22 moles o f g l u t a m i c a c i d , 9 moles o f g l y c i n e , 5 moles o f a l a n i n e , 7 m o l e s o f p r o l i n e , 17 m o l e s o f v a l i n e , 9 moles o f isoleucine, a n d 18 moles o f leucine, p e r m o l e o f e n t e r o t o x i n B.
A m i n o acid
No treatment*
A lkylation
A lkylation and performic acid oxidation
HzO ~ and extensive alhylation and performic acid oxidation
Lysine Histidine Ammonia Homoserine lactone Arginine Cysteic acid Methionine sulfoxides Aspartic acid Methionine sulfone Threonine Serine Homoserine Glutamic acid Proline S-Carboxymethyl homocysteine Glycine Alanine Half cystine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Methionine and related residues
33 5 29 o 5 o o 47 o 13 14 o
33 5 33 1.74 5 o o 47 0.64 13 14 --
33 5 4.80 5 1.84 o 46.6 o 13. I 14 --
33 4.53 43.16 1.27 5.13 1.92 o 46-85 6.59 12.66 14 -
22
22
21.92
22
7 o io 5 2 17 8 9 18 21 13 8
7 2.38 lO.85 5.77 trace 17 i .9 ° 9.93 17.oo 21 13 6.66
6.85 1.94 i i. i o 6.2o o 16. oo i .29 9.67 17.76 6.15 12.9 ° 8.03
6.76 o i 1.73 6.32 o 16.97 0.097 9. lO 17.45 12.63 12.6 i 7-95
* O b t a i n e d f r o m BERGDOLL et al. ~ a n d u n p u b l i s h e d o b s e r v a t i o n s o f H u a n g a n d B e r g d o l l of this l a b o r a t o r y .
w i t h respect to m e t h i o n i n e groups, the n o n l i n e a r i t y of the plot indicates t h a t n o t all the m e t h i o n i n e groups are e q u a l l y reactive. I t was a s s u m e d t h a t t h e r e are two classes of m e t h i o n i n e groups, i.e., " f a s t " a n d " s l o w " reacting in the enterotoxin. A b o u t 4 m e t h i o n i n e residues (50%) were a l k y l a t e d at a faster tate. The r a t e c o n s t a n t s for the fast reaction of modification a n d i n a c t i v a t i o n are 0.022 rain -1 a n d 0.043 m i n - L respectively. The "slow" r a t e c o n s t a n t s of modification a n d i n a c t i v a t i o n are 0.004 rain -1 a n d 0.003 rain -1, respectively. The precipitin reaction was diminished to less t h a n 15 % when all eight of the m e t h i o n i n e residues were modified.
Oxidation with H~O~ The reaction of h y d r o g e n peroxide with e n t e r o t o x i n B is shown in Fig. 3. T h e slow and fast r a t e c o n s t a n t s for o x i d a t i o n of m e t h i o n i n e in e n t e r o t o x i n B at p H 2.3 are 0.0023 min -1 a n d o.o21 min 1, respectively. A p p r o x i m a t e l y 6 m e t h i o n i n e residues are oxidized at a faster rate. T h e amino acid composition of an oxidized e n t e r o t o x i n B indicates t h a t n o t all the methionine residues are oxidized even after 12 h of reaction. Biochim. Biophys. Acta, 194 (1969) 2 7 9 - 2 8 6
283
MODIFICATION OF METHIONINE RESIDUES IN ENTEROTOXIN B 100
80 60 ~ e t
tt
~p
0 40 z_ 20
0.2
c
~
0.1
,
A olkylote
~ ~iiou
~
B
~
~
. IC
0
~o., l-
st' Met groups
J
Gz
,~oL °3 0.2
N Gt
0.1
i
I0 2T°
0
,
I 30 T U B E NUMBER
,
2
\
L
40
I
2
, i i E I P' I I ~ I I
34.
56 TIME
I 2 3 (HOURS)
5
6
Fig. I. C h r o m a t o g r a p h y of a l k y l a t e d e n t e r o t o x i n B on CM-cellulose. T h e elution p a t t e r n s were o b t a i n e d f r o m s a m p l e s w h i c h h a v e b e e n r e a c t e d w i t h iodoacetic acid for zero h o u r (top), 2.5 h (middle) a n d 5 h (bottom). S a m p l e after r e a c t i o n was directly t r a n s f e r r e d into a 0.9 c m X 3 ° c m CM-cellulose c o l u m n , e q u i l i b r a t e d w i t h o.oi M s o d i u m p h o s p h a t e buffer (pH 5.7). A f t e r w a s h i n g w i t h o.oi M s o d i u m p h o s p h a t e buffer (pH 5-7), to zero a b s o r b a n c e a t 277 m/z, stepwise elution w i t h o.05 M s o d i u m p h o s p h a t e buffer (pH 6.8) (Gt) a n d N a , H P O ~ (G,) (started a t arrow) was p e r f o r m e d . Flow rate, 5.4 m l / t u b e per 2 min. Fig. 2. S e m i l o g a r i t h m i c plot for t h e r e a c t i o n of iodoacetic acid w i t h e n t e r o t o x i n ]3. (A) Loss of m e t h i o n i n e d u r i n g modification. (B) Loss of a n t i g e n - a n t i b o d y r e a c t i o n d u r i n g t h e modification. T h e excess r e a g e n t s were r e m o v e d b y c h r o m a t o g r a p h y a n d dialysis. T w o classes of m e t h i o n i n e g r o u p s were a s s u m e d .
I00 80:
z -
-
B
A
BO 40
~.
Total Met groups
E. 2 0
~
,,=, 300 I"Slow"
~
¢-I0 J
#__ £ 5 ~
2
t
I
inactivation
st" inactivation
- groups
I
I
I
i
I 2 3 4 5 6 TIME
II
r
2
( HOURS )
z w
, " 2OC w
E
\"Fa~"Met
~
A
x
r
i
-,
r
.3
4
5
6
nca. IO0 z w I.-on.. 13_
t
c
F
I0
i
i
20 30 ,u.g E N T E R O T O X I N
i
40
Fig. 3. S e m i l o g a r i t h m i c plot for t h e r e a c t i o n of H~O 2 w i t h e n t e r o t o x i n B. (A) R e a c t i o n w h i c h w a s carried o u t a t p H 2. 3. (B) Loss of a n t i g e n - a n t i b o d y r e a c t i o n w h e n t h e r e a c t i o n was carried o u t a t p H 2.70. Fig. 4. Precipitin a n a l y s e s of e n t e r o t o x i n B a n d modified e n t e r o t o x i n . C u r v e A is a control sample. C u r v e s B - D s h o w a l k y l a t e d e n t e r o t o x i n 13 in w h i c h 2 (Curve B), 6 (Curve C), a n d 8 (Curve D) m e t h i o n i n e residues h a v e been modified, respectively.
Biochim. Biophys. Acta, 194 (1969) 279-286
284
F.s.
CHU, M. S. BERGDOLL
T A B L E II EFFECT
OF CHEMICAL
STAPHYLOCOCCAL
MODIFICATION
ENTEROTOKIN
OF THE
METHIONINE
RESIDUES
ON T H E
EMETIC
ACTIVITY
OF
B
Number of methionine residues modified
Methods of modification
Enterotoxin B (#g /monkey)
Activity*
None 6 8
None O x i d a t i o n b y H=O 2 A l k y l a t i o n b y iodoacetic acid
20 5° 5°
4-6]6 o/6 0/6
* N u m b e r of m o n k e y s s h o w i n g positive r e a c t i o n vs. n u m b e r of m o n k e y s challenged.
T a b l e I shows t h a t o n l y 7 m e t h i o n i n e residues were oxidized after t h e 12 h of reaction. A f t e r I h of reaction aggregation was o b s e r v e d when the solutions were neutralized. The rate c o n s t a n t for the i n a c t i v a t i o n of a n t i g e n - a n t i b o d y reaction is 0.020 min -1 when the reaction was carried out at p H 2.30. However, t h e r a t e c o n s t a n t s are 0.0006 min -1 a n d O.Ol 4 min -1 for t h e slow a n d fast i n a c t i v a t i o n when the r e a c t i o n was carried o u t at p H 2.7. Since the i n a c t i v a t i o n is m u c h slower at p H 2. 7 t h a n at p H 2.3 (rate c o n s t a n t s are o.ooo6 min -1 a n d O.Ol 4 min -1 for the slow a n d fast i n a c t i v a t i o n , respectively), t h e amino acid composition of the p r o d u c t o b t a i n e d at p H 2.7 has n o t been analyzed. 2.0
i
i
A
IOO
hJ ~1 .5
9O 80
70
iG°
LL 1.0
=_>
_j 5o
d
4o
B 30
0.5
# 2o 1.0 I
250
270
290
310
33,0
WAVELENGTH
350
1570 2,90
410
430
2
3
4
5
6
7
8
NO. OF METHIONINE RESIDUES MODIFIED PER MOLE OF ENTEROTOXIN B
(my.)
Fig. 5. Fluorescence s p e c t r a of e n t e r o t o x i n B a n d modified e n t e r o t o x i n B. C u r v e A r e p r e s e n t s t h e control sample. Curves B--D are a l k y l a t e d e n t e r o t o x i n B in w h i c h 2,5 a n d 8 residues of m e t h i o nine were modified. C u r v e E (dotted line) r e p r e s e n t s a n oxidized s a m p l e in w h i c h 5 residues of m e t h i o n i n e were oxidized b y H=O=. Fig. 6. Effect of chemical modification of m e t h i o n i n e residues on t h e a n t i g e n - a n t i b o d y reaction, a n d fluorescence i n t e n s i t y (335 m/~) of enterotoxin. [] a n d Ill, a n t i g e n - a n t i b o d y r e a c t i o n a n d fluorescence i n t e n s i t y of o x i d a t i o n p r o d u c t s , respectively. C) a n d 0 , a n t i g e n - a n t i b o d y reaction a n d fluorescence i n t e n s i t y of t h e a l k y l a t e d p r o d u c t s , respectively.
Biochim. Biophys. Acta, 194 (1 ~69) 279-286
MODIFICATION OF METHIONINE RESIDUES IN ENTEROTOXIN B
285
Effect of modification of methionine residues on the biological activity of enterotoxin B A typical precipitin curve of the modified and unmodified enterotoxin B is shown in Fig. 4. Modification of the methionine residues by either alkylation or oxidation resulted in a significant decrease of antigen-antibody reaction when more than 5 or 6 residues of methionine are chemically modified. The results of the monkey feeding tests are summarized in Table II. The emetic activity was lost when 6 or more residues of methionine were modified. Fig. 5 shows the fluorescence spectrum of the modified and unmodified enterotoxin B. The relationship between the decrease of antigen-antibody reaction and the quenching of fluorescence after oxidation or alkylation is presented in Fig. 6. Neither the antigen-antibody reaction nor t r y p t o p h a n fluorescence was decreased when a control sample was held at pH 2. 3 for 12 h. DISCUSSION
The experimental results indicate that methionine residues in enterotoxin B play a very important biochemical role. Amino acid composition analysis indicated that only methionine residues were altered after alkylation. The apparent loss of tyrosine residues was due to destruction by performic acid during analysis15, in because no loss of tyrosine residues was observed when the alkylated sample was hydrolyzed without performic acid oxidation. The amino acid analysis also suggested that the oxidation of methionine sulfur is the sole chemical modification of significancet hat occurred during the oxidation with H202 (last column of Table I). Although the disulfide bond might have been oxidized during the reaction, evidence has been obtained 1~,13 which indicates the disulfide bond in enterotoxin B is not essential for its biological activity. Kinetic analyses reveal at least 2 or 3 types of methionine residues in enterotoxin B. At approximately p H 2.7, 4 residues are probably located on the surface of the toxin. These 4 residues reacted with iodoacetic acid much faster at p H 2. 7 than the othersXV,TM and were almost instantly oxidized by the H,O, at p H 2. 3. Enterotoxin B m a y undergo a minor conformational change at pH 2. 3 which results in a faster reaction with HzO 2. No significant changes in the fluorescence properties and antigen-antibody reaction have been observed when 4 residues of methionine were oxidized. However, significant changes were observed when they were alkylated. This difference might be due to the alteration of the charges by the alkylation since the chromatographic behavior of the alkylated products has been significantly altered. Major conformational changes (loss of fluorescence) and loss of biological activity occurred when the 5th to 7th methionine residues were modified. These residues m a y play a predominant role in the structural and biological function of the enterotoxin. One methionine residue probably was buried inside of the molecule. Correlation of loss or decrease of antigen-antibody reaction with the change of conformation has been demonstrated in m a n y other proteins such as pepsin and pepsinogen 19,2° and hemoglobin and myoglobin 21 systems. The quenching of fluorescence in proteins after denaturation has been considered also to be a result of the conformational changes 2.. BOROFF AND FITZGERALD ~3 demonstrated the correlation between the loss of biological activity and the loss of tryptophan fluorescence of botulinum toxin. The correlation between the loss of antigen-antibody precipitin reaction and the quenching of fluorescence in the present study can be interpreted as a significant change of conformation. Since present knowledge about the mode of Biochim. Biophys. Acta, 194 (1969) 279-286
286
F . s . CHU, M. S. BERGDOLL
action of enterotoxin B and the conformation of the toxin in vitro remains unclear, whether the modified methionine residues are those on the active site is uncertain. However, from the observation of the complete restoration of the activity of the control samples which have been incubated at pH 2.3 for 12 h, and from the pH of the enterotoxin B receptor site which normally is slightly basic, it is clear that the inactivation is due to the conformational change rather than the effect on the active site. Enterotoxin B probably failed to fold to the original conformation after modification and thus lost its emetic activity. The conformational change may either render the susceptibility of enterotoxin B to enzymic digestion or alter the enterotoxin structure to a configuration which fails to interact with the target tissue. ACKNOWLEDGMENTS
The authors wish to acknowledge the technical assistance of Elizabeth Crary. This investigation was supported by public health service grant No. AI o7615 from the National Institute of Allergy and Infectious Diseases. REFERENCES I 2 3 4 5 6 7 8 9 IO ii 12 13 14 15 16 17 18 19 20 21 22 23
S. BERGDOLL, H. SUGIYAMA AND G. M. DACK, Arch. Biochem. Biophys., 85 (1959) 62. S. CHO, K. THADHANI, E. J. SCHANTZ AND M. S. BERGDOLL, Biochemistry, 5 (1966) 3281. R. BORJA AND M. S. BERGDOLL, Biochemistry, 6 (1967) 1467. M. AVENA AND M. S. BERGDOLL, Biochemistry, 6 (1967) 1474. S. BERGDOLL, in R. I. MATELES AND G. N. WOGAN, Biochemistry of Some Foodborne Microbial Toxins, M.I.T. Press, Cambridge, Mass., 1967, p. I. M. S. BERGDOLL, F. S. CHU, I.-Y. HUANG, C. ROWE AND T. SHIH, Arch. Biochem. Biophys., 112 (1965) lO4. E. J. SC~ANTZ, W. G. ROESSLER, J. WAGMAN, L. SPERO, D. A. DUNNERY AND M. S. BERGDOLL, Biochemistry, 4 (1965) IOli. L. SPERO, D. STEFANYE, P. I. BRECHER, H. M. JACOB¥, J. E. DALIDOWICZ AND E. J. SCIIANTZ. Biochemistry, 4 (1965) lO24. F. S. CHU, J. Biol. Chem., 243 (I968) 4342 . F. S. CHU, E. CRARY AND M. S. BERGDOLL, Biochemistry, 8 (1969) 2890. F. S. CHU AND E. CRARY, Biochim. Biophys. Acta, 194 (1969) 287. J. E. DALIDOWICZ, S. J. SILVERMAN, E. J. SCHANTZ, D. STEFANYE AND L. SPERO, Biochemistry, 5 (1966) 2375. I.-Y. HUANG AND M. S. BERGDOLL, Arch. Biochem. Biophys., 132 (1959) 423 . E. A. PETERSON AND H. A. SOBER, J. Am. Chem. Soe., 78 (1956 ) 751 . N. P. NEUMANN, S. MOORE AND W. I-I. STEIN, Biochemistry, I (1962) 68. C. H. W. HIRS, J. Biol. Chem., 2I 9 (1956) 611. W. J. RAY, JR. AND D. E. KOSHLAND, JR., Broohhaven Syrup. Biol., 13 (196o) 135. W. J. RAY, JR. AND D. E. KOSHLAND, JR., J. Biol. Chem., 236 (1961) 1973. H. VAN VUNAKIS, H. LEHRER, W. S. ALLISON AND L. LEVlNE, J. Gen. Physiol., 46 (1963) 589. J. F. GERSTEIN, H. VAN VUNAKIS AND L. LEVlNE, Biochemistry, 2 (1963) 964. M. REICHLIN, N[. HAY AND L. LEVlNE, Biochemistry, 2 (1963) 971. R. F. STEINER AND H. EDELHOCH, Nature, 192 (1961) 873. D. A. BOROFF AND J. E. FITZGERALD, Nature, 181 (1958) 751. M. F. C. R. M.
Biochim. Biophys. Acta, 194 (1969) 279-286