TRYPTOPHAN FLUORESCENCE IN HAIR 301 nearly 100%. The results presented in Table IV also include the analysis of hair that was fi rst reduced and subsequently re-oxidized with hydrogen peroxide. The data suggest a decrease in the fl uorescence intensity of Trp after treatment with hydrogen peroxide, as compared to reduced hair, to levels about 20% higher than the initial values obtained for untreated hair. Based on these data, one can conclude that the reduction–re-oxidation cycle does not reconstitute the original structure of untreated hair, at least as probed by Trp fl uorescence. In contrast to thioglycolates, which act as active reducing agents at pH 9, the use of 3% sodium hydroxide leads to a decrease in the intensity of Trp fl uorescence from 5.6 ± 0.3 ⋅105 cps to 2.4 ± 0.2 ⋅105 cps. The treatment of hair with high concentration alkalis such as 3% NaOH is employed in the process of relaxing curly, African hair. The chemistry of this process includes the reaction of HO− with disulfi des and subsequent formation of thiolate groups. Further transformations result in the formation of lanthionine groups and lysino- alanine residues. Disulfi de bond cleavage is signifi cant and was reported to be as high as 72% (20). In relaxed hair, disulfi des are replaced with lanthionine groups, which main- tain the stiffness and rigidity of the keratin matrix. A possible interpretation of these observations is suggested by reviewing the literature of protein model systems. One of them is a protein referred to as fusarium solani pisi cutinase, which is an enzyme with a single L-Trp, which is located close to a disulfi de bridge (21). It is also involved in a hydrogen bond with an alanine residue (22). According to Martinho et al. (23), there are both static and dynamic quenching mechanisms of the fl uores- cence of this Trp residue. Disruption of the Ala-Trp hydrogen bond, which occurs during melting of the protein, releases Trp from the viscinity of a cystine residue and results in a fourfold increase in Trp fl uorescence intensity. In addition, UV light, with wavelengths corresponding to the maximum of Trp absorption, breaks down the disulfi de bridge, re- sulting in a ten-fold increase in Trp fl uorescence quantum yield. The cleavage of the disulfi de bond increases the internal backbone mobility of the neighboring residues and releases the Trp residue from the viscinity of the cystine residue (24). Reduction of disul- fi des in the process of hair waving with thioglycolates has a similar effect. It also has a plasticizing effect on the amorphous matrix in cortical cells and increases the mobility of protein chains. Re-oxidation of cysteine residues into disulfi des, as a result of treatment with hydrogen peroxide, reconstitutes the network of disulfi de bridges and, consequently, brings about the reduction of Trp emission intensity. It should also be added that other Table IV Effect of Chemical Treatments on Trp Fluorescence of Dark Brown Hair Hair treatment Intensity of Trp fl uorescence at 336 nm × 105 (cps) Untreated Treated 6% NaTGA, pH 9.0 (15 min) 5.1 ± 0.2 7.8 ± 0.3 6% NaTGA (15 min)/2% H2O2 (5 min) 5.4 ± 0.2 6.4 ± 0.2 6% NH4TGA, pH 9.0 (15 min) 5.1 ± 0.2 8.4 ± 0.3 6% NH4TGA (15 min)/2% H2O2 (5 min) 5.3 ± 0.2 6.4 ± 0.3 3% NaOH (15 min) 5.6 ± 0.3 2.4 ± 0.2 Commercial perm (NH4TGA) (15 min) 5.2 ± 0.2 10.1 ± 0.5 Perm and neutralizer (15 min and 5 min) 5.2 ± 0.2 6.9 ± 0.2 The spectral analysis was carried out on dry hair at ambient conditions (40%–50% RH).

JOURNAL OF COSMETIC SCIENCE 302 amino acid side-chain groups, such as those corresponding to aspartate, glutamate, lysine, aspargine, and glutamine, can also quench Trp fl uorescence if located within 10 Å from the residue. This may also lead to decreases in Trp fl uorescence in hair containing a net- work of unreduced disulfi de bonds. EFFECT OF WATER CONTENT Figure 7 shows the fl uorescence spectra of brown hair with different levels of hydration. Completely dry hair, immediately after hair dryer application and cooled down to room temperature, displayed the lowest intensity of Trp emission at 5.9⋅105 cps at 336 nm. For completely wet hair the maximum emission was red-shifted to 343 nm and the intensity increased to 9.7⋅105 cps. For hair equilibrated at 50% RH, the observed emission posi- tion and its intensity were intermediate between dry and wet hair. It should also be pointed out that while the position and emission intensity of Trp fl uorescence is signifi - cantly affected by the content of water in hair, the peaks corresponding to kynurenines are not sensitive to such changes in the hydration of hair. This is evident from the fl uores- cence spectra shown in Figure 5 (excited at 290 nm) and was also confi rmed by spectra obtained by excitation at 320 nm, which showed no signifi cant change with a variation in the content of water. In addition, we also examined spectra of other types of hair, such as Piedmont and bleached, obtained at various levels of hair hydration. Qualitatively, the observed spectral changes were similar to those discussed above for brown hair. The effect of water on Trp emission in hair can be explained by hydrogen bond breaking during hydration involving Trp residues, similar to the effect of protein melting that leads to denaturation of cutinase by temperature increase (23). In this case the disulfi de bridges remain intact however, elimination of hydrogen bonds softens the keratin structure and/or helps to release Trp from the vicinity of cystine residues and induces the disrup- tion of Trp-disulfi de bridge complexes. Such structural transformations result in increased fl uorescence intensity in hair with high water content. Figure 7. Effect of water content on the fl uorescence spectra of brown hair.