Chemical alteration of human salivary proteins with 30% H₂O₂ solution

Objectives: Some salivary macromolecules penetrate enamel lesions and are adsorbed onto apatite crystal surfaces. Research has shown that removing salivary proteins from an enamel lesion with sodium hypochlorite solution enhances crystal growth. H₂O₂ at 20-35%, which is the active ingredient of in-office bleaching agents, denatures and/or degrades chromogenic molecules. We hypothesized that in-office bleaching can be used therapeutically to denature macromolecules present in enamel subsurface lesions and thereby to enhance remineralization. In this study, we support this hypothesis by demonstrating that exposure to 30% H₂O₂ solution chemically alters human salivary proteins.Methods: Five ml of resting whole saliva was divided into two halves. The H₂O₂ sample was mixed with isopropanol to a final concentration of 70% and centrifuged. We added 30% H₂O₂ to the precipitate, incubated it for 30 min, and then again added isopropanol to precipitate protein. Precipitate and supernatant were separated by centrifugation and analyzed by SDS-polyacrylamide gel electrophoresis with dithiothreitol. We used an identical procedure to prepare the H₂O sample except that we added H₂O instead of H₂O₂. Intact and fragmented molecules in H₂O₂ sample were examined by Coomassie Brilliant Blue (CBB) staining, Stains-All staining, and Western blotting.Result: Using CBB staining, we identified bands in the H₂O₂ sample that were not present in the H₂O sample. At the same time, certain bands in the H₂O₂ sample disappeared. Using Stains-All dye, we observed that the bands of acidic salivary mucin (> 250 kDa) were significantly larger in the H₂O₂ sample than in the H₂O sample. Using Western blotting with antibodies against serum albumin, we showed that such larger bands appeared as a result of reaction with anti-albumin antibody.Conclusion: Salivary proteins in resting whole saliva reacted with 30% H₂O₂ resulting in fragmentation of certain proteins and in forming mucin of a larger size, which contains albumin component.