442 SHORT COMMUNI'CATIONS, PRELIMINARY NOTES VOL. 15 (1954)

CORTICOTROPINS (ACTH)

V. T H E A P P L I C A T I O N OF O X I D A T I O N W I T H P E R I O D A T E TO T H E D E T E R M I N A T I O N

OF N - T E R M I N A L S E R I N E IN a - C O R T I C O T R O P I N

by

I R V I N G I. G E S C H W I N D AND CHOH HAO LI

Hormone Research Laboratory, University of California, Berheley, Calif. (U.S.A .)

A previous repor t from this labora tory 1 has emphasized the difficulty in obtaining quant i ta t ive yields of amino end groups from a-cort icotropin 2. Thus, the f luorodinitrobenzene (FDNB) method 3 yielded only o . i -o .2 moles of DNP-ser ine per mole, while the CS z procedure 4 yielded no 2-thio- 5-thiazolidone at alP. Application of EDMAN'S phenylisothiocyanate method 5 resulted in the recovery of circa 0.6 moles of serine phenyl th iohydan to in per mole of a-cort icotropin, calculated on the basis of light absorpt ion values at 270 111#; however, since both the ul traviolet absorpt ion curve and the results of ch roma tog raphy on paper revealed tha t the phenyl th iohydan to in obtained was part ial ly polymerized, the value given for recovery of the end group was only approximate . In a recent paper e, yields of 0.85 moles of serine phenyl th iohydan to in per mole of a-cort icotropin have been reported, using the EDMAN reaction carried out by means of the paper str ip technique ~.

The present communica t ion concerns the use of oxidat ion with periodate and the measurement of the formaldehyde formed thereby to determine the N-terminal serine of a-cort icotropin. The formation of formaldehyde is specific for N-terminal serine since a-cort icotropin contains nei ther hydroxylys ine nor carbohydra te . Oxidat ion with periodate has been employed previously for the determinat ion of N- terminal serine and threonine in the pept ides arising from acid hydrolysis of proteinsS, 9. More recently it has served to characterize the peptides obtained after saponification of the esters formed during acyl migrat ion in proteins 1°.

The details of bo th the oxidat ion with periodate and of the subsequent determinat ion of tile formaldehyde formed followed the recently published method of FRISELL et al. n, with slight modi- fications for the handl ing of smaller samples. Thus, only o. I ~ M of a peptide is employed and 0.025/~M

O/ of serine may be determined wi th an accuracy of ± 5 /o- The reliability of the method has been fur ther checked wi th the synthet ic dipeptides§, serylglycine and alanylserine. Quant i ta t ive recovery of formaldehyde was obtained from serylglycine, whereas alanylserine gave rise to no formaldehyde whatsoever under the condit ions of the exper iment .

With three different p repara t ions of a-cort icotropin 2 tr ichloracetate, the average recovery was 0.68 moles of formaldehyde per mole of hormone (Table I). Since a-cort icotropin t r ichloracetate is only part ial ly soluble at p H 7.5, in some exper iments the peptide was dissolved in 2 M urea to enable the oxidat ion to proceed in homogeneous solution, control exper iments indicating complete recovery of serine from urea solutions. In the presence of urea, however, the yield of formaldehyde from a-cort icotropin was unchanged. I t was fur ther demons t ra ted tha t shaking the solution during the oxidation, increasing the a m o u n t of periodate, or increasing the oxidat ion t ime also had no effect on the yield of formaldehyde.

T A B L E I

F O R M A L D E H Y D E F O R M E D F R O M T H E O X I D A T I O N O F ( 1 - C O R T I C O T R O P I N W I T H M E T A P E R I O D A T E

Preparation Yield o[ Formaldehyde/ Fovraaldekyde ~ a-corticotropin

No'* tam M/M

I 4 H D E o . i9 I o.77 4 H ' D o.I77 o.71 5 H ' D o. I42 o.57

Average 0.68

* 0.2 5/~M of a-cort icotropin was used in each exper iment . ** Each value is the average of duplicate analyses from two or more exper iments .

§ We are indebted to Dr. J. I. HARRIS for the gift of these peptides.

VOL. 15 (1954) SHORT COMMUNICATIONS, PRELIMINARY NOTES 443

On the basis of the well-known react ion of formaldehyde wi th proteins, it might be expected t ha t recovery of formaldehyde from a protein solution would no t be quant i ta t ive . To test this possi- bility, 0.25/~M of serine was added to o.25/~M of the hormone, and the solution was then subjected to oxidat ion wi th different quant i t ies of per iodate; de terminat ion of formaldehyde revealed t h a t in every case only 73 % of the added serine could be detected as the aldehyde. In other exper iments o.25/~M of the hormone was dissolved in a solution containing o.25/~M of formaldehyde. To this solution was added an a m o u n t of po tass ium iodate equivalent to the a m o u n t of sodium metaper iodate ordinari ly employed. The rest of the details followed the normal procedure. Under these conditions, only 31% of the added formaldehyde could be recovered. As expected, no formaldehyde was formed from a-cort icotropin itself when iodate was used. Fur the rmore , formaldehyde alone was quant i - ta t ively recovered.

If the percentage recovery of serine in the presence of a-cort icotropin is used as a correction factor (IOO/73), then the corrected a m o u n t of formaldehyde obtained from a-cort icotropin is 0.68 × lOO/73, or o.93 moles of serine per mole of hormone. On the o ther hand, if the calculation employs as a correction factor the recovery of formaldehyde in the iodate exper iment , a value of 0.68 × ioo/31 , or 2.2 moles of serine per mole of hormone is obtained. This lat ter value is much too high and reflects the fact t h a t recovery of formaldehyde in the presence of a-cort icotropin which has not been oxidized does not accurately reflect the reactions occurring during and following oxidat ion with metaperiodate . In a similar vein, it has been pointed out 11 tha t recovery of formaldehyde in the presence of cysteine is incomplete. However , if a solution of cysteine with added serine is t reated with metaperiodate , complete recovery of the serine as formaldehyde is observed.

The corrected value of 0.93 moles of N-terminal serine per mole of hormone compares very favorably with the uncorrected value of 0.85 moles per mole reported previously 6, and offers ad- ditional evidence for the identi ty of the N-terminal amino acid (serine) in a-cort icotropin.

The au thors wish to acknowledge the able technical assistance of EVELYN EMMRICH. This work was suppor ted in pa r t by grants from The Nat ional Ins t i tu tes of Health, U.S. Public Heal th Service (G 29o7), and from Eli Lilly and Co.

R E F E R E N C E S

1 A. L. LEvY AND C. H. LI, J. Biol. Chem., in press. C. H. LI, I. I. GESCHWIND, A. L. LEVY, J. I. HARRIS, J. S. DIxoN, •. G. PON AND J. O. PORATH, Nature, 173 (1954) 251; C. H. LI, I. I. GESCHWIND, J. S. DixoN, A. L. LEVY AND J. I. HARRIS, J. Biol. Chem., in press.

s F. SANGER, Biochem. J., 39 (1945) 507 . 4 j . LEONIS AND A. L. LEVY, Compt. rend. tray. Lab. Carlsberg, Sdr. chim., 29 (1954) 57- 5 p. EDMAN, Acta Chem. Scand., 4 (195 °) 283- 6 j . I. HARRIS AND C. H. LI, J. Am. Chem. Soc., 76 (1954) 3607. 7 H. FRAENKEL-CONRAT, J. Am. Chem. Soc., 76 (1954) 36o6. s A. H. GORDON, A. J. P. MARTIN AND R. L. M. SYNGE, Biochem. J., 35 (1941) I369. 9 p. DESNOELLE AND A. CASAL, Biochim. Biophys. Acta, 2 (1943) 64.

10 D. F. ELIOTT, in G. E. W. WOLSTENHOLME, Ed., The Chemical Structure o/ Proteins, J. and A. Churchill Ltd., London, 1953 .

11 W. R. FRISELL, L. A. MEECH AND C. G. MACKENZIE, J. Biol. Chem,, 2o 7 (1954) 7o9.

Received September 13th , 1954