Reversible reaction of ϵ-amino groups of cytochrome c with salicylaldehyde to produce cytochrome c polymers

BIOCHUMICA ET BIOPHYSICA ACTA

323

BBA 35178 R E V E R S I B L E REACTION OF e-AMINO GROUPS OF CYTOCHROME c W I T H S A L I C Y L A L D E H Y D E TO P R O D U C E CYTOCHROME c POLYMERS

J. N. WILLIAMS Jm AND R. M. JACOBS* Laboratory of Nutrition and Endocrinology, National Institute of Arthritis and Metabolic Diseases, National Institutes of Health, Bethesda, Md. (U.S.A .) (Received August 25th, 1967)

SUMMARY

I. Cytochrome c has been shown to react with salicylaldehyde at room temperature and p H 9.6 to give a cytochrome c-salicylaldehyde complex which is probably the azomethine derivative of the lysine e-amino groups and the aldehyde. 2. The reaction goes to completion, i.e., all 19 lysine residues react, when the ratio of aldehyde to cytochrome c in the reaction mixture is 19 to I or greater. 3. The fully reacted product is insoluble and completely unreactive in the NADH-cytochrome c reductase system from rat heart. 4. Dialysis of the reaction product removes the methinyl phenol groups completely. The cytochrome c product becomes soluble but is only 20% active in the NADH-cytochrome c reductase system. 5. Momentary treatment of the modified cytochrome (dialyzed reaction product) at p H 3 or I I completely restores its enzymatic activity. 6. Passage of the modified cytochrome through Amberlite CG-5o (Na +) indicated that at least 8 fractions were present. Further studies using Sephadex G-IOO columns standardized for determining molecular weights indicated that the monomer through hexadecamer polymers of cytochrome c were present.

INTRODUCTION

The e-amino groups of lysine in proteins have been treated with various reagents to modify the structure of the protein. Even with reversibly reactive trifluoracetyl derivatives 1 a high pH and lengthy hydrolysis are necessary to break the trifluoracetamide linkages. These conditions could result in irreversible conformational changes. A reaction between a protein and a reagent which takes place under very mild conditions and which is easily reversible could be useful for studying effects of structure and charge modification on properties of the protein. Such a reaction ~night be especially Present address: Division of Nutrition, Food and Drug Administration, Washington, D.C., U.S.A. *

Biochim. Biophys. Acla, 154 (I968) 323 331

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WILLIAMS JR, R. M. JACOBS

useful, for exalnple, to change the charge of the protein when removing groups of amino acids from the chain b y specific peptidases; the end-product can then be freed of the charge-changing groups with minimal effect on the conformation of the remaining polypeptide. A reaction of this type appeared to be possible by forming a azo-methine linkage between the e-amino groups of lysine in a protein and an aromatic aldehyde. Horse heart cytochrome c was chosen as a model for this study because of its high content of lysine ; and salicylaldehyde because of its solubility at a reasonable p H and inability to react beyond the formation of the simple Schiff's base. (Unsubstituted aliphatic aldehydes m a y react further because of the active methylene group adjacent to the aldehyde group.) A preliminary note on portions of this study has been presented earlier 2. METHODS

Reaction between cytochrome c and salicylaldehyde. Cytochronle c (horse heart, Sigma Type I I I , assay 98-1oo °/o pure) was allowed to react in 0.33 M K2HPO 4 (pH9.6) with salicylaldehyde (adjusted to p H 9.6 with KOH). The mixture was stirred at room temperature during the reaction. When the ratio of aldehyde to cytochrome c was 19 :I or greater, the reaction product was insoluble. Reversal of the azomethine reaction. Dialysis of the insoluble reaction product against 0.05 M K2HPO 4 (pH 9.6) at room temperature slowly removed the bound aldehyde, and the protein simultaneously went into solution. NADH-cytochrome c reductase assay. In a I-ml cuvette were mixed 55/*moles of sodium phosphate (pH 7.4), I / , m o l e of N A D H (pH 7.4), 5/,moles of KCN, and water to make o.9ml. In another cuvette were mixed 25 #moles of sodium phosphate (pH 7.4), 5/~moles of KCN, 0.3 ml of a I~o freshly prepared rat heart homogenate in o.I M sodium phosphate (pH 7-4), and water to make 0. 9 ml. At zero time, o.I ml of the cytochrome c preparation was added to both cuvettes. The cuvettes were mixed by inversion and the adsorbance of the second cuvette read against the first at 550 m# in a recording spectrophotometer for 2 rain to obtain rate of reduction, or after IO rain of standing at room temperature, to obtain the extent of reduction. In most cases the extent of reduction only was measured since there was good correlation between rate and extent of reduction under these conditions. The direct addition of salicyladehyde to the NADH-cytochrome c reductase system with native cytochrome c added separately had no effect on the activity under these conditions in the range of salicylaldehyde concentrations employed in the present study. Measurement of bound aldehyde. Bound salicylaldehyde was measured by treating the sample (plus water to make 2 ml) with 0.5 ml 0.04 M 2,4-dinitrophenylhydrazine in 2 M HC1 at 7 °0 for 30 rain, followed by addition of I ml of 2 M KOH. A blank and standard (o.I25/,mole of salicylaldehyde in I5/,1 ethanol) were treated similarly. After 3 rain the color was read at 560 m/, against the blank. RESULTS

Rate of reaction between cytochrome c and salic.vlaldeh3~de. A mixture of salicylaldehyde and cytochrome c in a molar ratio of IOO :I was allowed to react, and at IOrain intervals for I h, aliquots were removed. Reducibility by dithionite and effect of Biochim. Bioph3,s. Acta, 154 (19(o8) 323-331

CYTOCHROMEC POLYMERS

325

CO on the dithionite-reduced product were studied. Also the enzymatic activity of other aliquots in the NADH-cytochrome c reductase system were studied. In Fig. I it is seen that with dithionite alone a slight drop in reducibility of the product occurred during the first 30 rain with a slight rise thereafter. When CO was bubbled into the dithionite-reduced mixture for 3 min, the spectral m a x i m u m at 550 m# slowly approached the minimum at 535 m # until after I h CO completely flattened the spectrum. Activity of the product in the NADH-cytochrome c reductase system was lost rapidly and approached zero activity asymptotically after IO min. That the reactivity of the reduced product with CO and its activity in the NADH-cytochrome c reductase system indicate somewhat different effects on the cytochrome c molecule is shown by the quite marked difference in the shapes of the curves. 0.6

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When the level of aldehyde added to the reaction mixture was added directly to the NADH-cytochrome c reductase system using unmodified cytochrome c, no effect was obtained indicating that the effect of the aldehyde is upon the cytochrome c and not on the enzymesemployed in the assay system.

Effect of raHo of saHcylaldehyde to cytochrome c on CO reactivity of the product. When the aldehyde to protein ratio varied from zero to IOO and the reaction was allowed to proceed for 2 h (Fig. 2), CO reactivity of the dithionite-reduced product was first seen at a ratio of IO to I. The maximum effect occurred after a ratio of aldehyde to protein of 20 to I, or approximately when all 19 of the lysine e-amino groups of cytochrome c had reacted with saHcylaldehyde.

Effect of ra~io of salicflaldehyde ~o cytochrome c on solubility and NADH-cytochrome c reductasereactivi@ of the cflochrome c. Various levels of aldehyde were reacted with cytochrome c for I h and aliquots centrifuged at 2o00 x g for 15 rain. Cytochrome c was estimated in the precipitates and supe~atants. Activity of the reaction mixture Biochim. Biophys..4cta,

154 (1968) 323-331

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Fig. 3. Relationships between the ratio o f s a i i c y l & l d e h y d e t o c y t o c h r o m e c t o s o l u b i l i t y ~nd r e a c t i v i t y o f t h e P r o d u c t in the N A D H - c y t o c h r o m e c reduc~ase system. M e d i u m 0.33 M I ( 2 H P O ~ ; r o o m temperature; time o f re&ction bet~veen a l d e h y d e and protein I h; M i q u o t s s p u n at 2ooo X g f o r I 5 rain.

in the N A D H - c y t o c h r o m e c reductase s y s t e m was also measured. In Fig. 3 it can be seen that when the ratio of aldehyde to protein was IO to I a p p r o x i m a t e l y one-half of the cytochrome c became insoluble and was unable to react in the N A D H - c y t o chrome c reductase system. W h e n the ratio was increased to 19 to I, the protein became totally insoluble and almost completely unreactive in the N A D H - c y t o c h r o m e c reductase system. Again this indicates that the 19 lysine e-amino groups of cytochrome c react with salicylaldehyde; and when this occurs, the effects on the protein are Biochi~n. Biophys. Acla, ]54 (t968) 323 331

327

CYTOCHROME C POLYMERS

maximal. Whether, when the ratio of aldehyde to cytochrome c is IO to I, approximately one-half of the cytochrome c molecules in the reaction mixture have completely reacted with aldehyde and the remaining one half have not reacted at all, is a question that cannot be decided at present. Removal of methinylphenol groups from cytochrome c. When the insoluble cytochrome c-salicylaldehyde complex obtained from reacting a IOO to i ratio of aldehyde to protein was dialyzed against 0.05 M K2HPO 4 (pH 9.6) and aliquots removed at intervals during the dialysis, the results shown in Fig. 4 were obtained. Curve A shows that the aldehyde was removed rapidly during the dialysis with a temporary plateau after 14-15 methinyl phenol groups had been removed (from 5-7 h). Thereafter the loss of bound aldehyde proceeded as shown until all had been removed. Simultaneously from Cm've B it can be seen that when aliquots were removed during the dialysis and spun at 30 ooo rev./min for 30 rain the cytochrome c product became soluble with a plateau when about 35% had gone into solution. Thereafter, the protein became rapidly soluble. When aliquots during the dialysis were tested in the NADH-cytochrome c reductase assay system, they were found to be only 2o% active even when all aldehyde had been removed after 24 h.

Return of activity in the NADH-cytochrome c reductase system to the dialyzed preparation. It was found that if the 24-h dialyzed sample in Fig. 4 was brought to either p H 3 or i i for a few seconds and then neutralized to pH 7, its activity in the NADH-cytochrome c reductase system was completely returned. The effect of the p H 20~

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Fig. 4- R a t e of removal of salicylaldehyde from salicylalated c y t o c h r o m e c b y dialysis against 0.05 M K2HI~Oa. Curve A was obtained b y measuring b o t h aldehyde and c y t o c h r o m e c concent r a t i o n s in aliquots from t h e dialysis bag after various times of dialysis. Curve B was obtained b y spinning aliquots from the dialysis bag at 30 ooo × g for 3 ° min and measuring c y t o c h r o m e c c o n c e n t r a t i o n in the s u p e r n a t a n t s from the aliquots. Dialysis was carried out at room t e m p e r a t u r e . Fig. 5- Effect of p H on t h e r e a p p e a r a n c e of N A D H - c y t o c h r o m e c reductase activity in salicyla l d e h y d e - t r e a t e d - a n d - d i a l y z e d c y t o c h r o m e c. Aliquots of t h e dialyzed p r o d u c t were b r o u g h t to various p H values with acid or alkali addition for approx, io sec and t h e n r e t u r n e d at once to p H 7.

Biochim. Biophys. Acta, 154 (1968) 323-331

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J.N. WILLIAMS JR, R. M. JACOBS

a d j u s t m e n t on the 2o% active dialyzed p r e p a r a t i o n is shown in Fig. 5- These results are highly reminiscent of those of PAULa a n d MARGOLIASH AND LUSTGARTEN ~ on r e t u r n of a c t i v i t y to ' i n a c t i v e ' or polymeric cytochrome c. Separation of fractions from modified cytochrome c on Amberlite CG-5 o. As shown in Fig. 6, when the salicylaldehyde-reacted, 24-h dialyzed cytochrome c, was adsorbed on Amberlite CG-5o, prepared according to MARGOLIASH AND LUSTGARTEN4 a n d eluted with a NaC1 g r a d i e n t (o.21-o.76 M) at least 8 peaks were obtained. The peak labelled I ' in the figure is due to reduced cytochrome c. The considerable tailing after the s e v e n t h fraction indicates t h a t more fractions could be seen if better resolution were possible with this resin.

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Fig. 6. Separation on Amberlite CG-5o (Na+) of salicylaldehyde-treated-and-dialyzed cytochrome c. After adsorption of the product on the column, it was eluted with a NaC1 gradient (o.2i-o.7 6 M). Fig. 7. Comparison of reactivity of fractions 1-8 from the Amberlite CG-5o column in the NADFicytochrome c reductase (NCR) system and with CO as a function of the suspected average molecular weight of the fractions. In the lower part of the figure are the sedimentation coefficients of the fractions. W h e n the center cuts from each of these fractions were tested in the N A D H cytochrome c reductase system a n d with CO a n d the s e d i l n e n t a t i o n coefficients det e r m i n e d in the a n a l y t i c a l ultracentrifuge, the results shown in Fig. 7 were obtained. R e a c t i v i t y in the N A D H - c y t o c h r o m e c reductase system a n d with CO were s t u d i e d b y the m e t h o d s o u t l i n e d above. The results in the N A D H - c y t o c h r o m e c reductase system are calculated as the ratio of the total reducibility with dithionite less the total r e d u c i b i l i t y in the N A D H - c y t o c h r o m e c reductase system to the total reducibility with dithionite since the c o n c e n t r a t i o n s of cytochrome c in each of the fractions was different ; similarly, for the r e a c t i v i t y with CO. Thus a ratio of I would indicate complete n o n - r e a c t i v i t y in the N A D H - c y t o c h r o m e c reductase system a n d complete r e a c t i v i t y with CO. All three sets of results have been plotted against an abscissa c o m m o n l y used for representing s e d i m e n t a t i o n coefficients; i.e., (molecular weight) 2f3. I n this case it is assumed t h a t the fractions represent m o n o m e r or polymers of cytochrome c. The purpose of presenting all of the results as a f u n c t i o n of molecular weight is to a t t e m p t to u n c o v e r a relationship between each of the parameters and shape of the lnolecules. The s e d i m e n t a t i o n coefficient plotted versus (molecular weight) 2/3 should

Biochim. Biophys. Acta, 154 (1968) 323-331

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CYTOCHROME C POLYMERS

give a straight line for compact spheres. From the results it can be seen that, although fractions 2-8 react to the same extent (17%) in the NADH-cytochrome c reductase system, reactivity with CO is much less in all fractions. Moreover there appears to be a break in the curve for reactivity with CO after fraction 3. The shape of the curve for the sedimentation coefficients is quite similar to that for CO reactivity indicating perhaps a positive relationship between shape of the molecules and CO reactivity. The constant low reactivity of fractions 2-8 in the NADH-cytochrome c reductase system indicates that steric hindrance effects of one unit upon another are more important than shape in determining enzymatic reactivity of the fractions. Reactivity of the fractions with CO probably indicates a change in tertiary structure as the units aggregate, allowing entry of CO into units which are notably resistant to CO when entirely monomeric. However, this change is not very large as indicated by the relatively low reactivity with CO and by the facile reversibility of aggregation with acid or base to active monomer. Estimation of molecular weights of the products in the modified cytochrome c. The procedure of WHITAKER~using a 1. 5 cm × 19o cm Sephadex G-Ioo column equilibrated in I M NaCl-o.o2 M N a K PO~ (pH 7) was employed for estimating molecular weights of the various fractions obtainable from the modified cytochrome c. The column was standardized with blue dextran (for void volume) and yeast alcohol dehydrogenase, bovine serum albumin, ovalbumin, pepsin, a-chymotrypsin and cytochrome c monomer. The modified cytochrome c was obtained after treatment of cytochrome c with salicylaldehyde and dialysis for 24 h as in Fig. 4. The preparation was further dialyzed in I M NaCl-o.o2 M N a K POa (pH 7) for 24 h in the cold. Four ffmoles of modified cytochrome c were applied to the column and fractions collected in 2-in! portions. In Fig. 8 it can be seen that at least 8 fractions were readily resolved. Calculation of the molecular weights of each of these fractions indicated that they were indeed monomer through octamer based on a molecular weight for monomeric cytochrome c of 12 6oo O9 2 °.8

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Biochim. Biophys. Acta, 154 (1968) 323-331

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Since there was considerable tailing after the octamer, it was suspected that higher polymers were probably present. Fractions from the early effluent were combined and reapplied to a second standardized Sephadex G-ioo column. Results from this run are shown in Fig. 9. The fractional values were obtained because of the inaccuracies related to the poor resolution of this column. Although the resolution is by no means complete, the results indicate that polymers as large as the hexadecamer are probably present. DISCUSSION

et al.6,s

PALEUS AND NIELANDS7 a n d MARGOLIASH observed t h a t chrolnatog r a p h y of cytochrome c isolated from m a m m a l i a n tissues on weak cation exchangers, yields more t h a n one chromatographic species. I t was found t h a t these fractions, called Fractions I I b y MARGOLIASH el are m u c h less reactive in the cytochrome oxidase or succinate oxidase system t h a n is the n a t i v e protein. NOZAKI9 found t h a t a fraction of trichloroacetic acid-treated beef heart cytochrome c could be eluted from a cation exchanger at a higher ionic s t r e n g t h t h a n t h a t required to elute the n a t i v e protein. I t was observed t h a t the molecular weight of the fraction was double t h a t of the n a t i v e protein. MARGOLIASH AND LUSTGARTEN4 f o u n d t h a t t r e a t m e n t of monomeric cytochrome c witE 6o% ethanol or 5 % trichloroacetic acid at p H 4.5 produces a m i x t u r e c o n t a i n i n g monomer, dimer, trimer, and t e t r a m e r of cytochrome c. These polymers had little a c t i v i t y in the cytochrome oxidase system, b u t reacted with carbon monoxide. T h e y could be converted m a i n l y to m o n o m e r b y t r e a t m e n t with dilute acid or alkali. The yields of m o n o m e ~ t e t r a m e r prepared b y t r e a t m e n t with ethanol 4 were quite different from those in the present paper, however. Dimer p r e d o m i n a t e d considerably over m o n o m e r whereas the reverse was true for the fractions o b t a i n e d from dialysis of the cytochrome c salicylaldehyde complex. There was little i n d i c a t i o n also t h a t

al.

Biochim.Bioph3~s..4cla,154 (1968) 323

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CYTOCHROME c POLYMERS

polymers higher than the tetramer were produced by ethanol treatment. The present results indicate that polymers (dimer through hexadecamer) are produced by the salicylaldehyde-dialysis treatment. The reactivity of the polymers produced by MARGOLIASHin the cytochrome oxidase system and those reported here in the NADHcytochrome c reductase system appear to be quite similar. This is also true with respect to reactivity with CO. Thus the nature of the polymers produced appears to be quite similar whether produced by ethanol or salicylaldehyde with subsequent dialysis, but the mechanism of production with consequent elaboration of different proportions and numbers of polymers appears to be quite dissimilar. The point at which the polymers are produced in the present studies is unknown at present. Whether they are produced during the reaction of e-amino groups of cytochrome c with salicylaldehyde or during the subsequent dialysis cannot be answered. The insoluble nature of the salicylaldehyde complex precludes a separation of a series of salicylalated polymers. That the salicylalated cytochrome c differs considerably from the salicylalated and dialyzed cytochrome c is indicated by the total reactivity of the former with CO (Figs. I and 2) but the partial reactivity (maximum of I5-35 °/o) of fractions 2-8 in Fig. 7. Even though the salicylalated cytochrome c is completely reactive with CO, it is not irreversibly denatured. This is indicated by the fact that direct treatment of the insoluble complex with acid (data not reported here) or of the polymers obtained from the complex with acid or alkali completely restores the native cytochrome c activity.

ACKNOWLEDGEMENTS

The authors wish to thank Dr. WILLIAM CARROLLof the National Institute of Arthritis and Metabolic Diseases, National Institutes of Health, Bethesda, Md., for aid in determination of sedimentation coefficients in the analytical ultracentrifuge. REFERENCES I 2 3 4 5 6 7 8 9

R. F. GOLDBERGER AND C. ]~. ANFINSEN, Biochemist~, I (1962) 4Ol. J. N. WILLIAMS, JR. AND R. M. JACOBS, Biochem. Biophys. Res. Commun., 22 (1966) 695. ]~. G. PAUL, Acta Chem. Scan&, 5 (1951) 389 • E. MARGOLIASH AND J. LUSTGARTEN, J. Biol. Chem., 237 (I962) 3397. J. R. WHITAKER, Anal. Chem., 35 (1963) 195 °. E. MARGOLIASH, •. FROHWIRT AND E. WIENER, Biochem. J., 71 (1959) 559S. PALEUS AND J. B. •IELANDS, Acta Chem. Scan&, 4 (195 °) l°24. E. MARGOLIASH, Biochem. J., 56 (1954) 535. M. NOZAKI, J. Biochem. (Tokyo), 47 (196o) ,592.

Biochim. Biophys. Acta, 154 (1968) 323-331