Characterization of salivary esterase activity related to dental composite biodegradation

Background: The biodegradation of dental composites by human saliva is suspected to be mediated by esterases, however the exact nature of the enzyme(s) remains unknown. Objectives: To determine the effect of serine esterase inhibitor (phenylmethylsulfonyl fluoride (PMSF)) on the degradation of composite resin by human saliva and to characterize the enzymatic agents responsible for this activity. Methods: 1) Photopolymerized Z250 (3M Canada Ltd) composite samples were incubated (n=3) with saliva in the presence (0.5 mM) or absence of PMSF (37°C; pH 7.0) for 2 and 8 days. Incubation solutions were analyzed for BisGMA derived product (i.e. bishydroxypropoxyphenylpropane (BisHPPP)) using liquid chromatography. 2) Unstimulated human saliva samples were pooled, purified, and freeze-dried. Processed saliva samples (n=7) were reconstituted in and run through a gel filtration column (GFC). Fractions were assayed for esterase activity using para nitro-phenyl butyrate as a substrate. Fractions exhibiting the highest esterase activity were electrophoresed on polyacrilamide gels and bands of interest were digested for mass spectrometry analysis. Results: The addition of 0.5 mM PMSF to saliva-incubated groups showed a reduction in the relative amount of BisHPPP as compared to the non-inhibited saliva, down to 29.5 ± 5.1% and 38.7 ± 5.5% after 2 and 8 days respectively. GFC yielded a fraction with a peak esterase activity having a molecular weight of approximately 40 kD. The electrophoresis bands isolated within the fraction were identified as being albumin, amylase and carbonic anhydrase VI (CA-VI). The active site of the latter enzyme is reported to be highly conserved in CA-II, which shows esterase activity with respect to phenyl ester substrates. Conclusion: The inhibitory effect of PMSF on degradation by human saliva provides evidence that esterase activity (possibly CA-VI) has a key role in the biodegradation of composites. Acknowledgements: CIHR grant, 3M Canada Ltd.