Characterization of Peptides Released from Treatment of Type I Collagen with Hydrogen Peroxide

Abstract

Previous research has demonstrated that hydrogen peroxide is capable of hydrolyzing the peptide bond in proteins including collagen. Proteases, like collagenase, are also capable of hydrolyzing peptide bonds at specific amino acid residues producing fragments. The goal of this project was to characterize the size and nature of the peptides produced by treating proteins with hydrogen peroxide. In order to characterize the response of proteins in general to hydrogen peroxide, various albumin proteins were treated with varying concentrations, precipitated using trichloroacetic acid and centrifuged. The amount of peptide small enough to not be precipitated was measured in the supernatant using the Lowry assay. The results demonstrated that increasing concentrations of hydrogen peroxide resulted in increased amounts of peptides too small to be precipitated. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) was used to examine the size of these proteins following treatment with hydrogen peroxide treatment or the protease trypsin, which hydrolyzes following the amino acid residues arginine or lysine. Chick albumin showed a band at the expected MW of 43 kDa and treatment with hydrogen peroxide (5.0%) reduced the size slightly. When treated with trypsin, the original band as well and other bands were visible on SDS PAGE. These results suggest that hydrogen peroxide works differently than proteases to hydrolyze proteins. Since trichloroacetic acid cannot precipitate collagen, other methods were used to characterize the peptides released by hydrogen peroxide. Glycine, proline, and hydroxyproline, which are overrepresented in the collagen primary structure, were detectable using thin layer chromatography (TLC). Silica plates were spotted with 1 mg/ml solutions with 70%/30% (v/v) n‐propanol/water as mobile phase. Glycine produced a red spot with an retention factor (Rf ) of 0.37 while proline resulted in a yellow spot (Rf 0.37) and hydroxyproline was pink (Rf 0.46). When the tripeptide Gly‐Pro‐Ala was used, the spot was yellow with an Rf value of 0.18. This result suggests that amino acids and small peptides can be resolved using this method. Type I collagen was mixed with 6.7% hydrogen peroxide or collagenase or both or neither in dialysis tubing and the contents of the fluid outside the tubing was tested using this TLC method as well as by measuring hydroxyproline in a colorimetric test. Reverse phase high pressure liquid chromatography were used to measure Glycine and Proline,Support or Funding InformationAuthors gratefully acknowledge research and professional development funds from Stockton University.