Characterization of Effect of Hydrogen Peroxide on Hydrolysis of Collagen

Americans spend billions of dollars a year on teeth whitening and the active ingredient is usually hydrogen peroxide. The outermost layer of teeth is the enamel and the dentin is underneath: the major protein found in the dentin of teeth is Type I collagen. The goal of this project was to characterize the effect of hydrogen peroxide at concentrations similar to those used in over-the-counter-whitening strips on collagen. Type I collagen was mixed either with hydrogen peroxide, collagenase, both or neither and the amount of protein released was characterized in several ways: dialysis (with a molecular weight cutoff of 3.5 kDa) and gel filtration with an exclusion limit of 10 kDa. The amount of protein or peptide released was measured with a modified Lowry assay, which has been adapted to measure collagen accurately. Collagenase is a metalloprotease that hydrolyzes the peptide bond. When treated with collagenase, no protein was released but 17 mg of protein was released when treated with 7.5% hydrogen peroxide and 18 mg when treated with both. Similar results were obtained with collagen isolated from human teeth. The amounts increased as hydrogen peroxide concentrations increased. For the gel filtration column, untreated collagen emerged in the first fraction after the void volume but when pre-treated with hydrogen peroxide, there were proteins in later fractions. Type I collagen shows a molecular weight (MW) of 129 kDa when run on 6% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) that was silver-stained. This amount matches the expected value based on the subunits of the protein. The addition of hydrogen peroxide caused this band to disappear from the gel and no smaller MW bands were visible. When treated with collagenase, the original band as well as several smaller proteins of 80, 66 and 47 kDa are observed as collagenase produces the occasional hydrolysis with large protein fragments that result. No bands were observed when collagen was treated with both collagenase and hydrogen peroxide. These results suggest that hydrogen peroxide is capable of hydrolyzing type I collagen and produces peptides that are too small to be detected on the gels but have a size of 3.5 kDa or less. Support or Funding Information Authors thankfully recognize support by research and professional development funds from Stockton University.