278 Essays in Biochemistry 



tion in molecular weight unless the disulfide cross links have first been 

 cleaved. 18 Moreover, if hydrolysis by proteolytic enzymes is contem- 

 plated, rupture of disulfide bonds is frequently necessary before enzy- 

 matic attack can occur. Finally, Ryle and Sanger 23 have shown that 

 a mixture of peptides of the structure R — S — S— R' and R" — S — S — R'" 

 can rearrange easily to give R— S-S-R" + R'— S— S— R"'. It is 

 obviously desirable to rupture disulfide bonds and eliminate this pos- 

 sibility, inasmuch as the determination of the sequence of amino acids 

 in a long peptide chain already presents sufficient problems without 

 introducing rearrangements to complicate the picture still further! 



Two general methods for cleaving disulfide bonds exist, the oxidative 

 and the reductive. Sanger employed the performic acid oxidation pro- 

 cedure of Toennies and Homiller 21 in his work with insulin, by which 

 means each mole of cystine yields two moles of cysteic acid. Schram, 

 Moore, and Bigwood 25 and Mueller, Pierce, and du Vigneaud 26 have 

 described conditions under which the conversion is virtually quanti- 

 tative, and Hirs 27 has found that in ribonuclease methionine is also 

 quantitatively transformed to the sulfone. With the definition by 

 Mueller et al., 26 Hirs, 27 and Thompson, 28 of conditions under which 

 tyrosine is fully stable, the performic acid oxidation procedure appears 

 admirably suited for use with proteins, such as insulin and ribonuclease, 

 that contain no tryptophan. With the large majority of proteins that 

 contain tryptophan, however, difficulties will be encountered, for Wit- 

 kop and his co-workers 29 have shown that tryptophan is unstable to 

 oxidation. If a single product, such as formylkynurenine, could be 

 obtained in good yield after oxidation, the instability of tryptophan 

 would make little difference. Formylkynurenine containing peptides 

 would then be formed on partial hydrolysis, and total acid hydrolysis 

 of these peptides would yield kynurenine which should readily be 

 determined chromatographically. It seems likely, however, that oxida- 

 tion of a tryptophan-containing protein with performic acid, followed 

 by partial hydrolysis, would result in the formation of a number of 

 different fragments derived from each tryptophan residue, thus greatly 

 complicating subsequent structural work. 



The difficulty with tryptophan might be circumvented if the di- 

 sulfide bridges of a protein were cleaved by reduction instead of by 

 oxidation. Sodium in liquid ammonia or lithium borohydride (on the 

 unesterified protein) might be capable of reducing all the disulfide 

 bonds in the molecule without attacking other vulnerable groups (cf. 

 Roberts, 30 for example, and Bailey 31 ). It would then, of course, be 

 necessary to cover the newly formed sulfhydryl groups by some reagent 



