Modification of tyrosine during performic acid oxidation

440

SHORT COMMUNICATIONS, PRELIMINARY

NOTES

VOL.

15 (1954)

This analysis confirms the r e p o r t b y STEDMAN AND STEDMAN7 of a histone rich in lysine and relatively p o o r in arginine. The " f a s t " c o m p o n e n t is similar to GREGOIRE AND LIMOZlN'S 8 P histone which precipitated in the course of t h e i r p r e p a r a t i v e p r o c e d u r e : it is possible t h a t at the concent r a t i o n of e t h a n o l t h e y employed, a partial fractionation of the c o m p o n e n t s occurred. A l t h o u g h the " f a s t " c o m p o n e n t is electrophoretically simple, its b e h a v i o u r in the ultracentrifuge is very complex, a n d its h o m o g e n e i t y is accordingly uncertain. The studies on this fraction, in the ultracentrifuge, made b y Dr. K. V. SHOOTER will be r e p o r t e d in a later paper. Analyses of different p r e p a r a t i o n s of the " f a s t " c o m p o n e n t have s h o w n significant d e p a r t u r e s from the figures given above, b u t this m a y be due to the presence of impurities. B o t h isolated c o m p o n e n t s are o b t a i n e d in r a t h e r small yield and it is possible t h a t other p r o t e i n s m a y be p r e s e n t which migrate in electrophoresis w i t h one or o t h e r of t h e m . The a u t h o r s are indebted to Mr. D. W. F. JAMES for the electrophoretic e x a m i n a t i o n s mentioned. This investigation w a s s u p p o r t e d b y g r a n t s to the Chester B e a t t y Research I n s t i t u t e from the British E m p i r e Cancer Campaign, the J a n e Coffin Childs Memorial F u n d for Medical Research, the A n n a Fuller F u n d , and the N a t i o n a l Cancer I n s t i t u t e of the N a t i o n a l I n s t i t u t e s of Health, U.S. Public H e a l t h Service. REFERENCES 1 j . A. V. BUTLER, P. F. DAVISON, D. W. F. JAMES AND K. V. SHOOTER, Biochem. J., 57 (1954) xxiv. 2 p. F. DAVlSON, D. W. F. JAMES, K. V. SHOOTER AND J. A. V. BUTLER, Bioehim. Biophys. Acta, 15 (1954) 415 • 3 W. L. BLOOM, B. CODGELL AND G. T. LEWIS, Cancer Research, io (195 o) 205. 4 E. F. MCFARREN, Anal. Chem., 23 (1951) 168. s j . F. ROLAND AND A. M. GROSS, Anal. Chem., 26 (1954) 502. s F. G. FISCHER AND H. D6RFEL, Biochem. Z., 324 (1953) 544. 7 E. STEDMAN AND E. STEDMAN, P h i l Trans. B., 235 (1951) 565. 8 j . GREGOIRE AND M. LIMOZlN, Bull. Soc. Chim. Biol., 36 (1954) I. Received S e p t e m b e r I s t h , 1954

M O D I F I C A T I O N OF T Y R O S I N E D U R I N G P E R F O R M I C ACID O X I D A T I O N by E. O. P. T H O M P S O N

Biochemistry Unit, Wool Textile Research Laboratory, C.S.I.R.O., ]l~lelbourne (A uslralia) I n certain s t r u c t u r a l i n v e s t i g a t i o n s on p e p t i d e s and p r o t e i n s performic acid is used for the oxidation of disulphide and thiol s u l p h u r linkages to sulphonic acid groups. TOENNIES AND HOMILLER1 have s h o w n t h a t 88 % formic acid a n d 3 ° % h y d r o g e n peroxide (9: i v/v) react t o g e t h e r r a t h e r slowly to give a m a x i m u m c o n c e n t r a t i o n of perfornlic acid in 80 m i n u t e s at 26.5 °, and t h a t formic acid solutions of h y d r o g e n peroxide have a significant effect on only four free a m i n o acids, cysteine, cystine, methionine and t r y p t o p h a n . However, the addition of 3 ° % h y d r o g e n peroxide to a formic acid solution of insulin at r o o m t e m p e r a t u r e was found 2,3 to result in partial modification of tyrosine residues with f o r m a t i o n of two derivatives of u n k n o w n constitution, referred to as tyrosine X and tyrosine X X . A d i s a d v a n t a g e of this reaction is the a p p e a r a n c e in peptide c h r o m a t o g r a p h y 3 , 4 of at least two zones for each t y r o s i n e peptide (tyrosine X X was n o t f r e q u e n t l y encountered), t h e r e b y complicating the c h r o m a t o g r a m s and reducing the a m o u n t of peptide for identification purposes. Modification of tyrosine in insulin has been avoided by using only a slight excess of h y d r o g e n peroxide '~ a l t h o u g h s o m e w h a t lower yields of the sulphonic acid p e p t i d e s were t h e r e b y obtained. An a l t e r n a t i v e procedure, using excess oxidant, involves t r e a t m e n t of the p r o t e i n with preformed performic acid a t IO ° to - - I5 ° and r e m o v a l of the r e a g e n t by freeze drying. This has been successfully used on o x y t o c i n e and o t h e r compounds~,S, 9 w i t h o u t a p p a r e n t modification of tyrosine. I n our e x p e r i m e n t s tyrosine derivatives, glycyltyrosine and insulin h a v e been treated with performic acid solutions u n d e r a v a r i e t y of conditions and t y r o s i n e X and t y r o s i n e X X were recovered w h e n e v e r the c o n c e n t r a t i o n of h y d r o g e n peroxide was high. The two c o m p o u n d s have been identified as 3-chlorotyrosine a n d 3,5-dichlorotyrosine respectively. C h r o m a t o g r a p h y of glycyltyrosine which h a d been t r e a t e d w i t h 3 % h y d r o g e n peroxide in w a t e r or acetone gave only one zone. After e v a p o r a t i o n of the r e a g e n t hydrolysis w i t h hydrochloric acid gave glycine, tyrosine, t y r o s i n e X and tyrosine X X , w h e r e a s on hydrolysis w i t h sulphuric acid

VOL. 1 5

(1954)

SHORT COMMUNICATIONS, PRELI.MINARY NOTES

441

or b a r i u m h y d r o x i d e only glycine a n d t y r o s i n e were detected. P r e s u m a b l y traces of peroxide left behind on e v a p o r a t i o n of excess r e a g e n t react w i t h the HC1 used for hydrolysis to release chlorine which t h e n reacts w i t h the tyrosine. W h e n glycyltyrosine was oxidised w i t h performic acid r e a g e n t (preformed 3 ° m i n u t e s ) at - - 1 5 ° for 50 m i n u t e s , diluted w i t h an equal v o l u m e of w a t e r a n d excess r e a g e n t r e m o v e d by e v a p o r a t i o n in a r o t a r y e v a p o r a t o r 1° at 20-3 °0 t w o zones a p p e a r e d on p a p e r c h r o m a t o g r a m s . "With b u t a n o l acetic acid as solvent one s p o t R F o.2, gave glycine a n d t y r o s i n e on hydrolysis and a n o t h e r , R F 0.3, gave glycine and 3-chlorotyrosine w h e n h y d r o l y s e d w i t h sulphuric acid or b a r i u m hydroxide. The f o r m a t i o n of the chlorinated derivatives w i t h o u t t r e a t m e n t w i t h hydrochloric acid is p r o b a b l y due to chlorine p r o d u c e d d u r i n g c o n c e n t r a t i o n , from residual h y d r o g e n peroxide and chloride ion impurities in the reagents. If performic acid (preformed i h) w a s used for t r e a t m e n t of glycyltyrosine a t o ° or r o o m t e m p e r a t u r e and excess r e a g e n t r e m o v e d b y freeze d r y i n g no t y r o s i n e modification occurred. W h e n c h r o n l a t o g r a p h e d on D o w e x 5 ° c o l u m n s by the procedure of MOORE AND STEIN 1° w i t h p H 5 citrate buffer as eluent, tyrosine X emerged at 28-44 ml and t y r o s i n e X X at 39-59 ml. Tyrosine X and t y r o s i n e X X were isolated b y c o m b i n i n g the peak fractions and desalting on a small c o l u m n of D o w e x 5 o, in the acid form, b y d i s p l a c e m e n t w i t h I N a m m o n i a . The isolated tyrosine X gave a positive halogen test. 3-Chlorotyrosine and 3,5-dichlorotyrosine were p r e p a r e d b y chlorination of tyrosine. Chlorine generated f r o m calculated a m o u n t s of p o t a s s i u m p e r m a n g a n a t e and c o n c e n t r a t e d hydrochloric acid was carried b y a s t r e a m of c a r b o n dioxide into a 0.025 M solution of t y r o s i n e in glacial acetic acid containing 2 % 2 N hydrochloric acid at r o o m t e m p e r a t u r e . F o u r a t o m s of chlorine per mole of tyrosine gave b o t h 3-chloro- and 3,5-dichlorotyrosine. W i t h 8 a t o m s of chlorine per mole of t y r o s i n e only 3,5-dichlorotyrosine w a s obtained. T y r o s i n e X and t y r o s i n e X X m o v e d d u r i n g c h r o m a t o g r a p h y a t the same rate as 3-chloro- and 3,5-dichlorotyrosine respectively. I n b u t a n o l : acetic acid : w a t e r (4 : I :5 b y volume) on W h a t m a n No. I p a p e r R F values were t y r o s i n e 0.29, tyrosine X and 3-chlorotyrosine 0.39, t y r o s i n e X X and 3,5-dichlorotyrosine 0.50, valine 0.36, leucine o.5o. I n n - b u t a n o l : 2 N a m m o n i a ( i : I ) the distances m o v e d in 65 h were t y r o s i n e 13. 7 cm, t y r o s i n e X and 3-chlorotyrosine 5.6 cm, t y r o s i n e X X a n d 3,5-dichlorotyrosine 2.9 cm. These results indicate t h a t to p r e v e n t t y r o s i n e modification it is i m p o r t a n t to keep the h y d r o g e n peroxide c o n c e n t r a t i o n as low as possible at all times (i.e. use p r e f o r m e d performic acid reagent), to use materials free of chloride ions a n d to completely r e m o v e all u n r e a c t e d h y d r o g e n peroxide f r o m the p r e p a r a t i o n before t r e a t m e n t w i t h hydrochloric acid. I t is r e c o m m e n d e d t h a t the r e a g e n t should be p r e p a r e d from 3 ° % h y d r o g e n peroxide and 9 8 - 1 o o % performic acid, allowing t h e m to r e a c t t o g e t h e r for a t least i h at 20 ° before use. R e a c t i o n w i t h p r o t e i n m a y be effected at r o o m t e m p e r a t u r e if reaction t i m e s are n o t excessive a n d care is t a k e n to m o d e r a t e the e x o t h e r m i c reaction. A l t h o u g h cooling stabilizes performic acid it also r e t a r d s its f o r m a t i o n from unreacted h y d r o g e n peroxide. The m o s t i m p o r t a n t step is r e m o v a l of excess r e a g e n t and this is best effected b y freeze d r y i n g several times. F o r this p u r p o s e it m a y be necessary to dilute the m i x t u r e w i t h 5 io v o l u m e s of ice-water and quickly freeze, since formic acid t e n d s to melt d u r i n g freeze drying. R e m o v a l of excess r e a g e n t s from t h e liquid state, as in a r o t a r y e v a p o r a t o r , greatly increases the possibility of retaining small a m o u n t s of h y d r o g e n peroxide and t h e r e b y of modifying the tyrosine. W i t h the r e c o m m e n d e d p r o c e d u r e t y r o s i n e modification h a s been avoided w i t h insulin using t h i r t e e n fold excess r e a g e n t and reaction t i m e s of I h at 20 °, or 2 h a t 0% I t h a s also been applied to wool and to a keratin derivative 12 w i t h o u t f o r m a t i o n of chlorotyrosines. This work was c o m m e n c e d at the U n i v e r s i t y of U t a h , Salt Lake City, U.S.A. a n d t h e a u t h o r is i n d e b t e d to Professor EMIL L. SMITH for the t y r o s i n e derivatives used, his h o s p i t a l i t y and interest, a n d to Dr. J. M. SWAN of this l a b o r a t o r y for helpful discussions. REFERENCES 1 G. TOENNIES AND R . P. HOMILLER,

J. Am. Chem. Soc.,

64 (1942) 3054 .

2 F. SANGER, Biochem. J., 44 (1949) 126. 3 F. SANGER AND H. TUPPY, Biochem. J., 49 (1951) 463 • 4 F. SANGEE AND H. TuPP¥, Biochem. J., 49 (1951) 481. ti F . SANGER AND E . O. P. THOMPSON, Biochem. J., 53 (1953) 353e j . M. MUELLER, J. G. PIERCE, H. DAVOLL AND V. DU VIGNEAUD, J. Biol. Chem., 191 (1951) 3o9. 7 C. W. ROBERTS AND V. DO VIGNEAUD, J. Biol. Chem., 204 (1953) 87I. s C. RESSLER, S. TRIPPETT AND V. DU VIGNEAUD, J. Biol. Chem., 2o 4 (1953) 861. 9 C. W. H. HIRS, Federation Proc., 13 (1954) 23 TM 10 L. C. CRAIG, J. ,D. GREGORY AND W. HAUSMANN, Anal. Chem., 22 (195 o) 1462. 11 S. MOORE AND W. H. STEIN, J. Biol. Chem., 192 (1951) 663. 12 j . M. GILLESPIE AND F. G. LENNOX, Biochim. Biophys. Acta, 12 (1953) 481. Received S e p t e m b e r 2 I s t , 1954