Protein analysis of carious and sound dentine digested by pepsin

Objectives: Recently developed dentine caries removal systems based on chemo-mechanical or enzymatic principles are aimed at dissolving denatured collagen and other proteins in the lesion. The denatured collagen components in such dentine have not been extensively studied. The purpose of this study was to describe the protein profile of pepsin-digested carious and sound dentine using SDS-PAGE to find relevant markers for carious dentine. Methods: Carious and sound dentine powder was collected separately from 20 permanent teeth with a rose-bur on a low-speed hand-piece. 10 mg of carious and sound dentine powder respectively was decalcified using 10% EDTA at pH 7.4 for 48 hrs. The decalcified pellet was digested using pepsin (1,090 units per mg wet weight of pellet) at pH 2 under sequenced conditions: at 4C for 24 hrs, followed by 24 hrs at 23C and finally 24 hrs at 37C. After every 24 hrs, the soluble fraction was separated by centrifugation at 1,000g, and precipitated at a final concentration of 0.9M NaCl and analyzed in 15% SDS-PAGE. Results: For carious dentine, the soluble fraction after demineralization demonstrated 2 bands at 62 and 56 kDa ("caries bands"), not seen in sound dentine. Carious dentine digested at 4C revealed the same two bands. In sound dentine digests, two bands at higher molecular weight (100 kDa) represented the collagen alpha chains. As pepsin only digests the telopeptide of collagen at 4C, the "caries bands" probably represented denatured dentine collagen. In sound dentine, "caries bands" was seen only after digestion at 37C. They probably represented further breakdown of collagen as pepsin digests collagen non-specifically at 37C. Conclusion: The seemingly specific caries bands might be used as markers for carious dentine. They probably represent collagen that has been decomposed in a similar manner as pepsin digests native dentine collagen at 37C.