Relationship between collagen hydrolysate molecular

Transcription

1 j. Soc. Cosmet. Chem., 42, (January/February 1991) Relationship between collagen hydrolysate molecular weight and peptide substantivity to hair GILBERT R. MINTZ, GALE M. REINHART, and BRUCE LENT, CalBiochem Corporation, La Jolla, CA (G.R.M.), Revlon, Edison, NJ (G.M.R.), and VideoJet Systems, Inc., Chicago, il (B.L.). Received October Synopsis Cosmetic grade collagen hydrolysates were incubated with virgin (natural) and bleached/waved (damaged) hair tresses. The bound collagen peptides were removed by both a high-temperature (50øC) and a high-salt (0.5 M NaCI) soak. As much as four times more fluorescamine reactive and hydroxyproline-containing peptides are removed from damaged than from natural hair. Gel filtration shows differences in the molecular weight (MW) distribution of peptides present in the high-temperature and high-salt soakings. These results demonstrate two distinct types of proteins that bind to hair: high-molecular-weight (>30,000 daltons) and low-molecular-weight (1,000 to 3,000 daltons). The majority of the hydroxyproline-containing peptides at bind to bleached/waved hair is in the lower molecular weight range and is probably composed of more basic amino acids. INTRODUCTION Previou studies indicate that cosmetic grade protein hydrolysates are complex mixtures of various molecular weight (MW) polypeptides (1). The capacity of a substance to absorb/adsorb to a surface is referred to as substantivity. The substantive nature of collagen-derived peptides to human hair has been demonstrated (2-4). Typically, peptide substantivity has been shown by monitoring the presence of the amino acid hydroxyproline (which is a component of collagen but absent in hair) absorbed on hair after a series of water rinsings to remove non-specific peptides. Radiolabeling studies have demonstrated that small quantities of amino acids (5) and alkyl-dimethyl quaternary derivatives of hydrolyzed collagen peptides (4) are absorbed to hair. In all of the studies to date, no information is available on the selective removal and subsequent characterization of peptides from the complex hydrolysates applied to hair. Methods used to manufacture protein hydrolysates typically yield a broad MW distribution of peptides (1,000-30,000 daltons). Accordingly, it was of interesto characterize the collagen peptides that were removed from the protein hydrolysate solution by specifically binding to the hair. We have extended previous observations regarding the binding of collagen protein hydrolysates (peptides) to hair by characterizing the MW of the hydroxyproline-con- 35

2 36 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS taining peptide substantive to hair. Using two different soaking methods--high-temperature (50øC) and high-salt (0.5 M NaCl)--we have removed hydroxyproline-containing peptides bound to hair. Quantitative and qualitative differences in both peptide fractions were characterized using various analytical techniques. MATERIALS AND METHODS PRETREATMENT OF HAIR TRESSES Hair tresses of virgin brown DeMeo human hair were made in 7-inch lengths weighing approximately 3 grams. After the tresses were prepared and prior to treatment, they were washed by immersion in a 1% solution of Triton X-100 solution, rinsed thoroughly with warm tap water, and allowed to dry in a 48øC oven as described previously (6,7). Damaged hair tresses were prepared by immersing hair tresses in an alkaline solution of hydrogen peroxide at 32øC for 30 minutes. They were then washed and immersed for 20 minutes at room temperature in a 5% thioglycollate solution, water rinsed, and neutralized with a solution of 3% hydrogen peroxide by soaking for 12 minutes. Each hair tress was rinsed and allowed to dry. Both the virgin (natural) and bleached/waved (damaged) hair were washed with a 1% solution of Triton X-100 and dried as above before each treatment with protein. ABSORPTION AND REMOVAL OF PROTEIN/PEPTIDES FROM HAIR TRESSES Each hair tress (approximately 3 g) was soaked in a 5% (wt/vol) solution of collagen protein hydrolysate (LEXEIN X-250, a registered trademark for an aqueousolution of hydrolyzed collagen protein) for 10 minutes at 25øC. To remove nonspecifically adsorbed protein, the hair was rinsed with warm running tap water for 1 minute and squeezed to remove excess water. Removal of bound collagen peptides from the hair tresses was accomplished by sequential steps in the following solutions: 1) each swatch (3 g) was placed in 30 ml of deionized water and then put in a 50øC water bath for 45 min; 2) the hair swatches were subsequently soaked overnight at 25øC in 30 ml ofa 0.5 M NaCI solution to remove any remaining peptides. Both virgin and bleached/waved hair tresses were subjected to the high-temperature and 0.5 M NaCI soakings triplicate to compare variations from one tress to another. No experiments were done reversing the sequence of the soaking conditions. CONCENTRATION OF REMOVED PEPTIDES The separate fractions of high-temperature- and 0.5 M NaCl-removed peptides were concentrated by lyophilization. The dried powders were resuspended in deionized water to small volumes (1-2 ml) and stored in a freezer at - 4øC prior to further characterization and analysis. GEL FILTRATION The molecular weight distribution of peptides removed from the hair tresses was determined by standard gel filtration procedures on Sephadex G-50 and G-15 sizing columns. The columns were equilibrated in 0.15 M NaCI and 0.1% SDS, and fractions

3 COLLAGEN PEPTIDE SUBSTANTIVITY TO HAIR 37 of 1 ml were collected. The MW standards for the G-50 column were as follows: ovalbumin (43K), cytochrome C (12K), bradykinin (1,250) and glycyl-glycyl-glycine (171); and for the G-15 column: insulin B-chain (3,500), bradykinin (1,250), glycylglycyl-glycine (171). The void volumes and included volumes were determined with blue dextran, and phenol red or tyrosine, respectively, under identical column conditions. The presence of peptides (amino groups) at specific MW ranges was determined by fluorescamine labeling of the columns' fractions (1 ml) after chromatography of the peptides. Similarly, the MW distribution of peptides derived from collagen was determined by hydrolyzing select fractions (1 ml) and quantitating the hydroxyproline content (9). Approximately 1,000-1,500 relative fluorescent (RF) units of the high-temperature soaking and the 0.5 M NaCI soakings were chromatographed on both the G-50 and G-15 Sephadex columns. FLUORESCENT DERIVATIZATION OF PEPTIDES The presence of primary amine groups in the high-temperature and high-salt soaked fractions from the lyophilized and gel filtration columns was determined by fluorescamine reaction with samples accoming to previous procedures (10-13). The relative fluorescence was measured on a Gilford Fluoro IV fluorometer. The excitation and emission wavelengths were set at 390 nm and 480 nm, respectively. A freshly prepared solution of fluorescamine was used for all amino group derivatizations. HYDROXYPROLINE ASSAY Hydroxyproline assays of individual column fractions or of the substantive peptides removed from the hair tresses were determined by base hydrolysis of the collagen peptides followed by hydroxyproline analysis as described previously (8,9). Select column fractions were lyophilized to concentrate the material prior to hydrolysis and subsequent hydroxyproline analysis. The relative hydroxyproline content of the various peptide fractions was determined relative to a standard curve for hydroxyproline. Absorbance measurements for the quantitation of hydroxyproline levels were done on a Gilford Spectrophotometer Model 260. To determine the background absorbance (hair tress never treated with the protein hydrolysate) and demonstrate that no hydroxyproline remained on hair tressesoaked in high temperature and 0.5 M NaCI, samples of hair tresses (10 mg) before and after the protein hydrolysate were hydrolyzed and assayed for hydroxyproline. MATERIALS Fluorescamine and all other reagents were obtained from Sigma Chemical. The lyophilizer was a Virtis Model Bench Top 3 from American Scientific Products. Sephadex G-50 and G-15 were purchased from Pharmacia. RESULTS AND CONCLUSIONS REMOVAL OF PEPTIDES FROM HAIR Virgin (natural) and bleached/waved (damaged) hair tresses were incubated with a cos-

4 38 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS metic grade collagen hydrolysate (X-250) (washed to remove unbound non-specific peptides) and subsequently treated at high temperature and high salt to remove specific bound peptides. Quantitative and qualitative analyses of fluorescamine-reactive amino groups released from hair tresses in both the high-temperature and high-salt fractions were determined. It should be noted that throughout these experiments we did not change the sequence of peptide soaking (i.e., high salt followed by high temperature). As shown in Table I, relatively low levels of fluorescamine-reactive amino groups were released by high temperature and high salt from both virgin and bleached/waved hair that had never seen the collagen peptides hydrolysate (5% wt/vol). In contrast, in hair tresses (virgin and bleached/waved) treated with collagen peptides, we observed a fold higher level of removed fluorescamine-reactive amino groups per weight of hair (3 g) in both the high-temperature and high-salt soaked fractions. These results suggest that most of the fluorescamine-reactive material removed from hair (i.e., both virgin and bleached/waved) treated with the cosmetic protein hydrolysate was due to the release of collagen peptides. Furthermore, in both the high-temperature and highsalt soakings, we observed twofold more fluorescamine-reactive amino groups released from bleached/waved hair than from virgin hair in tresses treated with the collagen hydrolysate. These results are consistent with previous observations that less collagen peptide is bound to virgin than bleached/waved hair as determined by quantitative hydroxyproline analysis of intact hair samples (1-3). Because it was not possible to radiolabel individual peptides in the protein hydrolysate prior to incubation with the hair tresses, we were unable to accurately quantirate the amount of peptide removed from the 5% protein/peptide solution or the exact amount of peptide removed from the hair during both soaking methods. Accordingly, comparative hydroxyproline analyses of the various fractions were used as a qualitative determination for the confirmation that collagen peptides from the protein hydrolysate were bound and subsequently removed from the hair tresses. HYDROXYPROLINE ASSAY OF REMOVED PEPTIDES To further demonstrate that the fluorescamine-reactive peptides removed from hair by Table I Fluorescent Quantitation of Amino Groups in the Removed Fractions From Hair Tresses Removed fraction High-temperature removal Virgin (- protein hydrolysate) Virgin (q- protein hydrolysate) Bleached/waved (- protein hydrolysate) Bleached/waved (q- protein hydrolysate) 0.5 M NaCI removal Virgin (- protein hydrolysate) Virgin (q- protein hydrolysate) Bleached/waved (- protein hydrolysate) Bleached/waved (q- protein hydrolysate) Total RF/Wt hair tress O.O92 2.O3O O O RF ---- relative fluorescence. Each measurement represents an average of two hair tresses. All fluorescent measurements were done in duplicate. Each hair tress weighed at least 3.0 grams.

5 COLLAGEN PEPTIDE SUBSTANTIVITY TO HAIR 39 both high temperature and high salt were derived from collagen, aliquots of each fraction were hydrolyzed and assayed for hydroxyproline. As shown in Table II, no hydroxyproline was detectable after both soaking conditions on virgin or bleached/waved hair tresses not treated with the collagen hydrolysate. It is generally known that hair (keratin) protein does not contain hydroxyproline (14). In contrast, hair tresses treated with the collagen hydrolysate and subjected to both soaking conditions had hydroxyproline-containing peptides in each fraction (see Table II). As indicated in Table II, about twice as much hydroxyproline-containing peptides were released from virgin hair in the high-temperature soaking than from the high-salt soaking, whereas, in the bleached/waved hair, about fourfold higher levels of hydroxyproline were present in the high-temperature soaking fractions than in the high-salt conditions. Similar results were obtained with different preparations of protein hydrolysate. Our observation support earlier results that bleached/waved hair binds more hydroxyproline-containing peptides than virgin hair (1,2,6). Furthermore, the total hydroxyproline released from the same weight of hair in both soakings was approximately the amount observed in previous work quantitating hydroxyproline-containing peptides bound to hair (2). In separat experiments, we assayed base-hydrolyzed samples of hair tresses for residual hydroxyproline after both the high-temperature and high-salt soakings. The results demonstrate that no detectable hydroxyproline remained on the hair, implying that the soaking conditions were sufficiento remove quantitatively all of the bound hydroxyproline-containing collagen hydrolysate (see Table II). MW CHARACTERIZATION OF PEPTIDES REMOVED FROM BLEACHED/WAVED HAIR To further characterize the substantive collagen peptides removed from hair tresses, we first chromatographed the intact solution of the collagen hydrolysate on a Sephadex G-50 column prior to incubation with the hair tress. As shown in Figure 1A, there was a wide distribution of peptides between MWs of 1,000-30,000, as indicated by fluorescent labeling of the peptide amino groups in select fractions. Also shown are select Table Quantitation of Hydroxyproline in Peptide Fractions Removed From Hair Tresses II Hydroxyproline (txg) Removed peptides Hair tress Temp. 0.5 M NaC1 Virgin (- protein hydrolysate) Bleached/waved (- protein hydrolysate) Virgin (+ protein hydrolysate) Bleached/waved (+ protein hydrolysate) Bleached/waved hair tress treated with protein hydrolysate, washed/removed N.D. N.D. N.D. N.D g 25 g g 4O g N.D. N.D. = not detected. The hydroxyproline assay was done on the concentrated and lyophilized fractions as described in Materials and Methods. Each point represents an average of duplicates two different concentrations and quantified relative to a standard curve for hydroxyproline. Each hair tress was approximately 3.0 grams.

6 ._ 40 JOURNAL OF THE SOCIETY OF COSMETI CHEMISTS o x o x 9o Fraction No. Figure 1. Elution profile from Sephadex G-50. A) Protein hydrolysate solution; B) Heat-extracted hydrolysate from bleached/waved hair; C) High-salt-extracted hydrolysate from bleach/waved hair. Protein extraction and detection as described in Materials and Methods. Fluorescamine-reactive amino groups ( -- ) and hydroxyproline (& - &) were monitored as described. Arrows ( ' ) in Part C indicate location of fractions with low levels of hydroxyproline.

7 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, suggesting 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 substantivity 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 another that doesn't react well with fluorescamine but is relatively high in hydroxyproline content. Additional evidence that the majority of the 1ow-MW hydroxyproline-containing 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 separat experiments, the high-salt-extracted peptides removed from damaged hair

8 42 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS were chromatographed on a G-50 column as shown in Figure 1C. In contrasto the high-temperature-removed peptides, a relatively high content of high-mw fluorescamine-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 the high-salt-removed fraction (see Table II), the G-50 column fractions were assayed for the presence of this amino acid. Preliminary characterization of the hydroxyproline content of the peptide(s) present in select column fractions 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 distribution 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 fraction 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 removed from hair tresses with high salt contain very low quantities of both 1ow-MW (<500 daltons) fiuorescamine-detectable peptides, and 1ow-MW hydroxyproline-conraining peptides. These results extend previous studies on the binding of hydroxyproline-containing peptides 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 distinct 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-

9 ,_ COLLAGEN PEPTIDE SUBSTANTIVITY TO HAIR 43 BO V o,i, Vi,I, 2.0 o e ß ß el e ß All ß ß ß ß B ß 0.6 5O 4O 0.4 o 3O 20 ß ''- '' '. 0 % ' ß..... A- A-- A-- A-- A-- A -- e A' A--I'A- - l e_ e _ _A e_ e.e Ae_ e _, øo Fraction No. Figure 2. Elution pattern from Sephadex G-15 of collagen hydrolysate peptides extracted from damaged hair. A) High-temperature or B) High-salt extraction. Monitored fluorescamine ( -- ) and hydroxyproline (A -- A) content of protein fractions placed on a G-15 column. Relative fluorescence and hydroxypro- line content in select fractions were monitored as described in Materials and Methods. ture swells the hair structure, allowing exit of adsorbed peptides from the hair shaft, whereas the high salt removes collagen peptides bound via ionic interactions to the hair. ACKNOWLEDGMENTS We acknowledge the work of Dave Simnick in earlier studies related to peptide substantivity to hair. We thank Hank Weightman and Ron Caesar for preliminary amino acid analysis of the peptide fractions. We also thank Barbara Berk for assistance in preparation of this manuscript. REFERENCES (1) E. S. Stern and V. L. Johnsen, Studies on the molecular weight distribution of cosmetic protein hydrolysates, J. Soc. Cosmet. Chem., 28, (1977).

10 44 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS (2) E. S. Stern, Studies on the substantivity of collagen-derived polypeptides to human hair, Inolex Technical Report 17 (1984). (3) E. S. Cooperman and V. L. Johnsen, Penetration of protein hyrolysates into human hair strands, Cosmetics and Perfumery, 88, (1973). (4) R. T. Jones and C. A. Brown. The behavior of cationic cellulose derivatives containing fatty quat groups, Int. J. Cosmet. Science, 10, (1988). (5) J. K. Herd and R. H. Marriott, The sorption of amino acids from shampoos onto hair, J. Soc. Cosmet. Chem., 10, (1959). (6) V. L. Johnsen, Innovation in protein and technology, Cosmet. Toiletr. 92, (1977). (7) S. A. Karjala, A. Karler, and J. E. Williamson, The effect ofph on the sorption of collagen-derived peptides by hair, J. Soc. Cosmet. Chem., 18, (1967). (8) S. A. Karjala, J. E. Williamson, and A. Karler, Studies on the substantivity of collagen-derived polypeptides to human hair, J. Soc. Cosmet. Chem., 17, (1966). (9) G. Huszar, J. Maiocco, and F. Naftolin, Monitoring of collagen and collagen fragments in chromatography of protein mixtures, Anal. Blochem., 97, (1980). (10) S. Udenfriend, S. Stein, P. Bohlen, W. Dairman, W. Leimgruber, and M. Weigele, Fluorescamine: A reagent for assay of amino acids, peptides, proteins and primary amines in the picamole range, Science, 178, (1972). (11) N. Nakai, C. Y. Lai, and B. L. Horecker, Use of fluorescamine the chromatographic analysis of peptides from proteins, Anal. Biochem., 58, (1974). (12) M. Weigele, J. F. Blount, J.P. Pengi, R. C. Czaijkowki, and W. Leimgruber, The fluorogenic ninhydrin reaction. Structure of the fluorescent principle, J. Amer. Chem. Soc., 94, (1972). (13) M. Weigele, S. I. DeBernardo, J.P. Pengi, and W. Leimgruber, A novel reagent for the fluorometric assay of primary amines,j. Amer. Chem. Soc., 94, (1972). (14) P. Flesch, Chemical data on human epidermal keratinization and differentiation, J. Invest. Dermatol., 31, (1958).