236 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS Table III Properties of N-AIk¾1 Maleamic and N-Alk¾1 Maleimides Maleamic Acid • Maleimide • Derivative Melting Point, øC Melting Point, øC Hexyl 78 ø 125 ø @ 5.5 mm a Heptyl 75-77 ø 43-44 ø Dodecyl -- 56-59 ø Benzyl 140-142 ø 73-75 ø aBoiling point. bObserved me[ting points were within the range reported by Coleman et al (7). REDUCTION, % 60 1'20 TIME, min. Figure 2. Effect of time on reduction of hair by 0.1 M DTT at self-pH, 35 C In using the next higher homolog, i.e., N-heptyl maleimide, the alkylation reaction could also be completed within 2 h. Following the alkylation with N-ethyl maleimide, disulfide bonds without producing any byproducts such as mixed disulfides. Its efficac'r: as a reductant allowed us to perform the reduction swiftly at neutral pH and a tempera, i) ture of 35øC. ß Hair samples were treated with unbuffered solutions of 0.1 M DTT (self-pH, 5.4) 25:1 bath ratio, 35øC for various times. Levels of reduction were calculated on the basis': of the SH content determined via mersalyl acid titration (9). These data agreed with the reduction levels determined from residual disulfide analyses (via amino acid:i analysis) after cyanoethylation of the free SH groups. A plot of the reduction levels against time is shown in Fig. 2. 5. Alkylation of hair with N-alkyl maleimides.' N-hexyl maleimide was used to alkylate reduced hair containing 0.6 m moles of SH per gram of hair. The extent of reaction was monitored by determining the residual SH following the alkylation. Under the condi-: tions employed, the alkylation was complete within 2 h. The alkylation was carried out in 20 per cent n-propanol/0.04 M phosphate buffer, pI-I 7 at 35øC, 100:1 bath ratio under these conditions the extent of hydrolysis of the N-alkyl male imides is negligible.

HYDROPHOBIC BONDS AND HAIR T1LEATMENTS Table IV Effect of Time on the Alkylation of Hair with N-Ethyl and N-Hexyl Maleimide '• 237 Time, Minutes Degree of Alkylarion, Per Cent N-Ethyl Maleimide N-Hexyl Maleimide 30 75 82 60 81 94 120 86 100 .i •[Maleimide] = 0.01 M Solvent = 20 per cent n-propanog0.04 M phosphate (pH 7) buffer temperature ½ 35øC bath Ratio = 100:1. approximately 15 per cent of the SH remained unreacted after 2 h, while a cor- responding sample of hair treated with the N-hexyl maleimide contained no residual SH (Table IV). This finding was in accord with the data presented by Heitz (6), who determined the second-order rate constants for the binding of N-heptyl maleimide to i::i.i yeast alcohol dehydrogenase. There, it was shown that the reaction rate for the N- ß heptyl maleimide reaction was approximately 8.4 times that observed with N-ethyl :'•': maleimide. This was somewhat unexpected, since a chainlength effect was not ob- ::: served in the reactions of these maleimides with cysteine and glutathione (6). We ob- served similar enhancement of the rate of alkylation in the case of reduced hair. .:'.:'::: Following reduction with thioglycolic acid, the sample was rinsed only briefly prior to the alkylation, and thus, residual thioglycolic acid remained in the fiber. At the end of a 24-h treatment, the cysteinyl residues were completely blocked by N-heptyl maleimide, while free thioglycolic acid was still detected in the alkylating solution. This observation and the previous results on the alkylation of reduced keratin indicate that •: an increase in the alkyl sidechain of the maleimide leads to faster rates of alkylation in spite of the .unfavorable diffusion factor. Such an enhancement in the reactivity may be '•i tentatively ascribed to the interaction between the alkyl sidechain of the reactant and the nonpolar residues of the keratin, which apparently provide an effective hydro- phobic environment for the combined cystine. .. •., 6. Swelling properties of the alkylated hair.' The extent of internal modification of ke- ,:.•:.. ratin often can be readily assessed from the change in the swelling characteristics of this : protein. Thus, fission of the disulfide bonds is accompanied by an increase in water •, imbibition, which is almost directly proportional to the number of crosslinks severed. According to our hypothesis of hydrophobic modification of hair, the introduction of , apolar residues should compensate for at least some of the disulfide bond breakdown. A strong support for this view was obtained from the liquid retention measurements of reduced and reduced-alkylated hair (Table V). The reduction treatment alone causes a large increase in swelling in both water and aqueous alcohol. Alkylation of reduced hair with methyl iodide or N-ethyl maleimide slightly intensifies the swelling. This incre- ment in hydration is probably caused by the elimination of weak hydrogen bonding involving the sulfur hydrogen and the apparent inability of the methyl or N-ethyl deri- vatives to establish any specific interactions with the environment. On the other hand, alkylation with either N-hexyl or N-heptyl maleimides brings about significant decrease in hydration. Obviously, the introduction ofhydrophobic residues can impart