Detection and activity of peroxidase in the in situ formed enamel pellicle

Abstract

Aim Peroxidase is the main salivary antioxidant. The aim of the present study was to detect and to characterise peroxidase in the in situ enamel pellicle. Methods Bovine enamel slabs were fixed on maxillary splints and carried by six subjects for different times (3, 30 and 120 min) on buccal and palatal sites. Pellicle bound peroxidase activity was determined fluorimetrically using 2′,7′-dichlorofluorescin as a substrate. The peroxidase molecules present in the pellicle were visualised with the gold-immunolabelling technique and evaluated by TEM. Furthermore, effects of polyphenols and hydrogen peroxide on peroxidase and its enzymatic activity were examined. Results All pellicles which were tested revealed peroxidase activity and labelled peroxidase molecules were detected in all samples. The numbers of gold-labelled peroxidase molecules detectable in cross-sections of the pellicles were correlated significantly with the pellicle formation time. After 3 min, 0.50 ± 1.01 labelled molecules were detected (30 min: 1.42 ± 1.98; 120 min: 4.15 ± 4.13, ANOVA, p < 0.001). The mean immobilised peroxidase activity exposed at the surface amounted to 24.4 ± 27.7 mU/cm 2; no continuous increase of activity with formation time was found. Hydrogen peroxide and polyphenolic beverages inactivated peroxidase activity of the pellicle. Despite these inhibiting effects, considerable amounts of peroxidase molecules were still detectable by gold-immunolabelling. After contact with the inhibiting agents in situ, peroxidase activity of the pellicle reconstituted slowly. Conclusion Peroxidase activity is present in the pellicle already after 3 min of formation time, but is inhibited by the substrate and polyphenolic beverages.