COLLAGEN PEPTIDE SUBSTANTIVITY TO HAIR 41 column fractions assayed for hydroxyproline. The results indicate that throughout most of the MW range the hydroxyproline level parallels the relative fluorescence however, in the low MW range (1,000-2,000), where the concentration of fluorescent detectable peptides decreases, the concentration of hydroxyproline-containing peptides actually increases. The highest level of hydroxyproline-containing peptides chromatograph in a range where the fluorescamine-reactive peptides are low in MW and relatively low in concentration. In preliminary experiments, we rechromatographed the 5 % (wt/vol) solution of collagen protein hydrolysate that had been incubated with 3 g of hair tress. The results indicate a diminution of fluorescence-reactive peptides in the 1,000-3,000-MW range, sug- gesting a selective removal of these peptides but not a complete removal of the collagen peptide(s) from this MW range, since only 3-g hair tresses were used in the substanti- vity experiments (data not shown). We have demonstrated that hydroxyproline is present in both the high-temperature and high-salt soaking fractions from hair tresses treated with collagen peptides (see Table II). Accordingly, it was of interest to determine the MW distribution of both fractions of removed peptides relative to the whole cosmetic grade collagen protein hydrolysate. As shown in Figure lB, fluorescamine-reactive peptides in the high-temperature-removed fraction of damaged hair chromatographed in a relatively sharp peak near 2,000-3,000 MW. To identify where the collagen peptides chromatographed, we hydrolyzed the peptides in select column fractions and assayed for the presence of hydroxyproline. The highest concentration of hydroxyproline-containing peptides was present at a slightly lower MW (i.e., 1,000) than the peak of fluorescamine-reactive peptides (3,000). This suggests that there may be different peptides in the collagen hydrolysate, with widely varying levels of hydroxyproline. Furthermore, it is generally known that proline and hydroxyproline as secondary amines escape detection with fluorescamine under certain reaction conditions (12, 13). However, the presence of hydroxyproline in the peptides fractionated on the G-50 column demonstrates that the high-temperature-removed peptides were derived from the collagen hydrolysate applied to the hair. The results in Figure lB demonstrate that there are at least two broad classes of peptides that bind to bleached/waved hair: one containing low levels of hydroxyproline and an- other that doesn't react well with fluorescamine but is relatively high in hydroxyproline content. Additional evidence that the majority of the 1ow-MW hydroxyproline-con- taining peptides and fluorescamine-labeled peaks are derived from collagen comes from our observations that very little fluorescamine-reactive amino groups (2.5%, see Table I) are removed from bleached/waved hair tresses that never saw the collagen hydrolysate. The selective removal and subsequent binding of 1ow-MW peptides (1,000-3,000 daltons) from a complex mixture of cosmetic grade collagen peptides suggests that MW is an important characteristic for peptide binding to bleached/waved hair. Furthermore, control bleached/waved hair tresses (not treated with the protein hydrolysate) soaked and chromatographed on a G-50 column have a fluorescamine-reactive peak in this 1ow-MW range (1,000-3,000 daltons) that comprises less than 5% of the peak RF of the peptides removed by high temperature from bleached/waved hair. In separate experiments, the high-salt-extracted peptides removed from damaged hair

42 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS were chromatographed on a G-50 column as shown in Figure 1C. In contrast to the high-temperature-removed peptides, a relatively high content of high-MW fluoresca- mine-reactive peptides were present at the void volume of the column (30,000 MW). The lower MW, high-salt-removed peptides were much broader in MW distribution than the high-temperature-removed peptides (see Figure lB,C). Despite the relatively low level of hydroxyproline in the high-salt-removed fraction (see Table II), the G-50 column fractions were assayed for the presence of this amino acid. Preliminary charac- terization of the hydroxyproline content of the peptide(s) present in select column frac- tions indicates that low levels of this amino acid were present at both the 30,000- MW and 1,000-MW positions (see arrows, Figure 1C). Because the high-salt fraction contained lower levels of both fluorescamine-reactive peptides and hydroxyproline, a more definitive characterization will require isolation of larger quantities of material. The results in Figure 1 demonstrate the selective binding of peptides of more defined MWs to hair tresses from a complex mixture of a cosmetic grade collagen hydrolysate. Furthermore, the qualitative differences in the MW profiles of the two different methods for removing bound peptides from hair are consistent with the idea that the final MW of the collagen hydrolysates is an important criterion for peptide binding to bleached/waved hair. It should be noted that preliminary observations suggest a similar peptide MW distri- bution for fluorescamine-reactive collagen peptides removed from virgin hair treated with protein hydrolysate (data not shown). CHARACTERIZATION OF LOWER MW HYDROXYPROLINE-CONTAINING PEPTIDES Qualitative differences in the MW distribution of the high-temperature and high-salt peptide(s) soakings were more apparent when we fractioned each sample from damaged hair on Sephadex G-15. As shown in Figure 2A, the high-temperature-removed frac- tion has a broad distribution of fiuorescamine-detectable peptides between 200-1,500 MW. In contrast, when the 0.5 M NaCl-removed fraction was chromatographed on G-15 (see Figure 2B), only high-MW peptides (1,000) were detected. When both column fractions were assayed for the presence of hydroxyproline, we observed very distinct patterns (see Figure 2A,B). The high-temperature-removed peptides contain a much higher content of 1ow-MW peptides (di- and tri-peptides) with a relatively high content of hydroxyproline. In contrast, within our detection limits, the peptides re- moved from hair tresses with high salt contain very low quantities of both 1ow-MW (500 daltons) fiuorescamine-detectable peptides, and 1ow-MW hydroxyproline-con- raining peptides. These results extend previous studies on the binding of hydroxyproline-containing pep- tides to virgin and bleached/waved hair by demonstrating the selective adsorption of peptide(s) via size (MW) and presumed charge to hair keratin. In the current study using a cosmetic grade collagen hydrolysate, it is clear that there are at least two dis- tinct populations of collagen peptides that bind to the hair: high-MW (30,000) and 1ow-MW (1,000-3,000). The majority of the hydroxyproline-containing peptides that bind to bleached/waved hair is in the lower MW range. Previous results demonstrate that adsorption of collagen hydrolysates to hair keratin increases with decreasing MW of the peptides (1). We presume that the high tempera-