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Clinical Oral Investigations
Published in: Clinical Oral Investigations 1/2009

01-03-2009 | Original Article

Characterisation of lysozyme activity in the in situ pellicle using a fluorimetric assay

Authors: Christian Hannig, Bettina Spitzmüller, Matthias Hannig

Published in: Clinical Oral Investigations | Issue 1/2009

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Abstract

Lysozyme is among the most protective enzymes in the pellicle layer. The aim of the present study was to establish a precise fluorimetric assay for determination and characterisation of lysozyme activity immobilised in the initial in situ formed pellicle. For in situ pellicle formation, bovine enamel slabs were fixed on maxillary splints and carried by six subjects for different times (3, 30 min) on buccal and palatal sites. The enzymatic assay was based on hydrolysis of cell walls from Micrococcus lysodeicticus linked to a fluorogenic substance. When the substrate is hydrolysed, a fluorescing product is released. Furthermore, the effects of chlorhexidine and black tea on lysozyme in the in situ pellicle were investigated. The fluorimetric method allowed direct determination of the enzyme activity with the slab inside the well of a microtiter plate. The mean immobilised activity over all samples amounted to 68.67 ± 27.35 U/cm2 (desorbed activity = 46.76 ± 21.18 U/cm2). The enzyme activity exposed at the pellicles’ surfaces increased in a time-dependant manner and showed a Michaelis–Menten kinetic. Chlorhexidine and black tea reduced lysozyme activity of the in situ pellicle significantly. After rinsing with tea or chlorhexidine, V max was reduced, whereas K m remained unaffected indicating a negative allosteric effect of the V type. The fluorimetric method is appropriate for determination of pellicle lysozyme activity. The influence of effectors on immobilised lysozyme activity can be monitored.
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Metadata
Title
Characterisation of lysozyme activity in the in situ pellicle using a fluorimetric assay
Authors
Christian Hannig
Bettina Spitzmüller
Matthias Hannig
Publication date
01-03-2009
Publisher
Springer-Verlag
Published in
Clinical Oral Investigations / Issue 1/2009
Print ISSN: 1432-6981
Electronic ISSN: 1436-3771
DOI
https://doi.org/10.1007/s00784-008-0213-9

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