PROGRESS IN THE CHEMISTRY OF DISULFIDES 305 about 13 per cent/-cystine in addition to qualitatively possessing the same amino acids. These proteins differ in the sequence in which the amino acids occur along the peptide chains. The proteins most likely differ in the identity of the amino acid joined to the cystine peptide link at the site of the disulfide cross link. In the case of insulin, Sanger (5) has shown that one important sequence involving cystine is the tri-peptide unit--glycine- cystine-alanine-. In the case of keratin the large amounts of dicarboxylic acids present as aspartic and glutamic acids lead us to believe that in a good number of chains several of these dicarboxylic acids are joined to cystine at the site of the crosslink. There occurs in nature a tripepride, glutathione, which consists of glutamyl-cystyl-glycine. It has been established by Phillips and co-workers (55) that about 25 per cent of the combined cystine in wool keratin is resistant to the attack of chemical reagents such as sulfite, alkali, cyanide, permanganate, etc. This resistant portion has been designated as the D fraction. Reed and others (56) have reported that commercial alkaline thioglycolate solu- tions effectively reduce only about 75 per cent of the cystine present in hair. The remaining resistant fraction seems to correspond to the D fraction observed in wool. Studies of peptides derived from wool hydrolyzates reveal that there is a juxtaposition of glutamic and cystine residues (57). The presence of the bulky side chain group of the former amino acid residue may well serve as an effective barrier to chemical attack upon the disulfide bond. Benesch and Benesch (58) attribute the greater resistance ofgluta- thione to oxidation as compared with cysteine as being due to the glutamyl residue with which the sulfur can hydrogen bond. If this proves to be the case then certain disulfide linkages in hair would be shielded by this ex- ternal steric effect and hence may account for the D fraction. Aside from the academic interest in the external barrier effect, what are the practical consequences of this phenomenon ? It might well be that the essential success of the hair waving process is due largely to these few resist- ant bonds which make up the D fraction. These resistant disulfide link- ages serve to maintain the skeletal structure of the hair when it is treated with reagents that cleave the other disulfide links. If these resistant links were not present, application of a reducing agent would then cleave all crosslinks and thus produce a material capable of undergoing plastic flow. Hence the hair will have lost its fiber properties. Severely damaged hair may well be characterized by a destruction of a good number of these resist- ant groups. It may now be seen that the danger inherent in employment of too strong a thioglycolate solution or too long a processing time is that all crosslinking disulfide bonds will be broken, yielding a plastic non-fibrous material. It is thus not necessary and, in fact, potentially detrimental to cleave all S--S bonds. Just enough bonds of the non-resistant, non-skeletally important

306 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS type should be cleaved so as to make the hair pliable and permit it to as- sume the shape of the curlet. This essentially is the advantage of using a more controllable reducing agent, such as the mercaptan employed in cold waving, rather than the harsh reducing agents such as sodium meta bi- sulfite which cleave SS bonds indiscriminately. Even partial reduction of resistant SS bonds accompanied by parallel chain stoppage or increased lateral chain separation raises the unhappy specter that an isolated partly shielded mercaptan group may be formed within the hair. In the subse- quent oxidation step customarily employed to convert the mercaptan to a disulfide crosslink either of the two following situations may arise: 1. The W--SH group finds itself as an isolated entity incapable of re- linking with any neighboring mercaptan group. Subsequent attack by a strong oxidizing agent will succeed in oxidizing this mercaptan to combined cysteic acid. As a result the hair as a fiber is now intrinsically that much weaker for having lost a formerly resistant disulfide linkage, or 2. The W--SH group might be too isolated to allow for its immediate oxidation. In such a case the remaining more favorably situated ruercap- tan groups are converted to disulfide links and this isolated mercaptan is sealed into the interior of the hair. With the passage of time and the continual kinetic chain slippage and folding, this mercaptan group will find itself within the immediate proximity of a disulfide crosslinkage. At such time the distinct possibility of disulfide-mercaptan interchange arises. Owing to the ease with which this interchange (59) can occur, this isolated mercaptan will be capable of acting as an "internal plasticizer," resulting in a slow exchange of S--S and SH bonds along the protein chain and producing stress relaxation or loss of the permanent wave. This effect is similar to what one observes when thiokol rubber under stress is subjected to mercaptan vapor (60). duVigneaud (61) has suggested that a similar mechanism may be responsible for the progressive gradual inactivation of insulin which occurs on the addition of small amounts of cystine to a solu- tion of this protein. On the basis of the results of our "test tube" experiments we cannot, of course, explain all the subtilities of the hair waving process. What we have tried to show in the last section of this article, however, is how infor- mation obtained from these new studies on disulfide cleavage can be used to explain the over-all chemistry of the hair waving process. We hope that our research may help to rationalize the empirical chemical procedures which are now employed in hair waving and to suggest new and more effec- tive methods for the waving of hair. REFERENCES (1) duVigneaud, V., "A Trail of Research in Sulphur Chemistry and Metabolism," Ithaca, Cornell University Press (1952). (2) Calvin, M., Chem. Eng. News, 31, 1735 (1953). (3) Wald, G., and Brown, P. K., 7- Gert. PhydoL, 35, 797 (1952).