Immobilisation and activity of human alpha-amylase in the acquired enamel pellicle

Abstract

Amylase is an important salivary component and structural element of the acquired enamel pellicle. Aim of the study was to establish a method for precise and direct determination of pellicle bound amylase activity in order to analyse kinetics and activity of the immobilised enzyme. Six bovine enamel slabs (5mm diameter) were fixed on individual maxillary trays and worn by five subjects for different times (3, 30 and 120 min) on buccal and palatal sites on different days. Slabs were removed from the trays and rinsed with aqua dest. Afterwards, pellicle bound amylase activity was determined directly with a photometric method using 2-chloro-4-nitrophenyl-4-O-beta-D-galactopyranosylmaltotriosid (GalG2CNP) as substrate yielding the coloured product chloronitrophenolate (CNP). All investigated pellicles exhibited immobilised amylase activity. Mean activity was 1.39 +/- 187 mU/cm(2) (n=87, range 0.14-11.5 mU/cm(2)). Product formation of CNP by immobilised amylase was linear over time. Pellicle bound amylase showed a Michaelis type kinetic (Km = 3.3 x 10(-3) M). Immobilised activity on buccal surfaces ranged between 0.25 and 11.1 mU/cm(2) (palatal slabs: 0.14-3.06 mU/cm(2)). Thirty minutes pellicles formed on buccal sites exhibited significantly higher immobilised amylase activity (2.85 +/- 3.65 mU/cm(2)) than palatal ones (0.63 +/- 0.32 mU/cm(2)). Amylase activity showed great intraindividual variability when comparing same positions on different days.

Conclusion: Pellicle bound amylase activity can be determined directly with GalG2CNP and shows a Michaelis Menten kinetic. Enzyme activity of the amylase immobilised in the in situ pellicle reveals great intra- and interindividual differences.