HAIR AND HYDROLYZED WHEAT PROTEINS 195 added to a stirred solution of the hydrolyzed wheat protein (20 ml 10% solution 2.06% active) at pH 7.5 over a period of 30 minutes at ambient temperature. The reaction mixture was stirred for 1 hour. Hydroxylamine (1 ml 1.5 M pH 8.5 Analar grade) was then added and stirring was continued for a further one hour. This solution was then purified using Sephadex G15 resin (Sigma-Aldrich) gel filtration media. The fluores- cently labeled hydrolyzed wheat protein to be purified was placed onto a column of the Sephadex G15 resin (1000 mm x 25 ram), previously equilibrated with phosphate buffer (0.05 M pH 7.0 containing 0.1 M sodium chloride), and eluted through the column using the same phosphate buffer (300 ml) at a flow rate of 1 ml/min. Fractions (10 ml) were collected and analyzed using size-exclusion HPLC (HP 1100 Tosohaas TSK G2000SWxl column, phosphate buffer eluent [0.05 M pH 7.0 containing 0.1 M sodium chloride], 25øC, UV detection at 220 nm, 0.6 ml/min flow rate). The fractions containing the purified fiuorescently labeled hydrolyzed wheat protein were combined and concentrated by rotary evaporation to approximately 10% total solids and subse- quently analyzed by size-exclusion HPLC to ensure that purity was maintained. ANALYSIS OF THE FLUORESCENTLY LABELED HYDROLYZED WHEAT PROTEIN SOLUTION It was essential that, prior to treating hair, the fiuorescently labeled hydrolyzed wheat protein was pure and free from any contamination with unreacted FITC or FITC de- composition products. Accordingly, a number of experiments and analyses were carried out to confirm that the Sephadex G15 gel filtration resin was capable of effectively separating the various components of the reaction mix. Size-exclusion HPLC (as de- scribed above) was used to characterize the various components of the reaction mixture. In particular, it was used to establish the purity of the fluorescently labeled hydrolyzed wheat protein eluted from the Sephadex column. The following solutions were exam- ined: A. Hydrolyzed wheat protein. B. Control FITC (FITC put through the above reaction procedure, but with the protein replaced by water). C. FITC-hydrolyzed wheat protein reaction mix prior to purification. D. Purified fluorescently labeled hydrolyzed wheat protein. The results in Figure i show that the hydrolyzed wheat protein (A) and the fiuorescently labeled hydrolyzed wheat protein (C,D) both have a peak maximum at an elution time of approximately 20 minutes. The unreacted FITC (B) has a peak maximum at an elution time of approximately 35 minutes. The polydispersity of the chromatograms is low, and the elution times are sufficiently different, such that it is possible to differentiate the various fractions obtained from the Sephadex column during purification. The relevant fractions were collected and the pure fiuorescently labeled hydrolyzed wheat protein was separated from the reaction mix as shown in Figure 1D. The purified fluorescently labeled hydrolyzed wheat protein was analyzed and found to contain total solids (9.3%), ash (7.6%) and thus, by difference, an active labeled hy- drolyzed wheat protein content of 1.7%. It was diluted to 1% active content before being used to treat hair. The following types of hair were chosen for treatment with the purified FITC-labeled peptides:

196 JOURNAL OF COSMETIC SCIENCE 30 25 ß • 20 • lO 5 0 20 30 40 50 I Tkne (minutes) Figure 1A 2.5 2 1.5 0.5 0 -0.5 10 20 30 Tkne (m•utes) Figure lB 1.8 1.6 1.4 1.2 0.8 0.6 0.4 0.2 0.0 -0.2 0 10 20 30 40 5o T•e (mhutes) o lO 20 3O Tkne (minutes) Figure 1D Figure 1C Figure 1. Size-exclusion HPLC chromatograms. A. Initial hydrolyzed wheat protein B. FITC control. C. FITC-labeled peptides before purification. D. Purified FITC-labeled peptides. 1. Untreated root-ends--brown Caucasian. 2. Split ends--blonde Caucasian. 3. Bleached--root-end brown Caucasian (using commercially available bleaching kit [Wella Hair Streaking Kit] two treatments given). 4. Permanently waved--root-end brown Caucasian (using commercially available perm treatment [Alberto VO5 select perre] two treatments given). 5. Relaxer treated--root-end brown Caucasian (hair relaxer RS-4-30-1 [formulated by Croda Oleochemicals] containing 2% sodium hydroxide used as a single treatment). 6. Relaxer treated--ethnic black (as in 5, but hair was physically straightened during treatment). Hair types were supplied by De Meo Brothers, New York, and International Hair Importers and Products Inc., New York. Swatches (made up of 30 x 4 cm hair fibers) of each of the above were treated with aqueous solutions of the fluorescently labeled hydrolyzed wheat protein for 30 minutes or overnight (16 hours), rinsed (in 2 x 250 ml water, each rinse lasting 10 seconds),