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

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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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Acknowledgement

The study was supported by a grant from the DFG (Deutsche Forschungsgemeinschaft, no. HA 5192/1-2, no. HA 2718/3–3). The authors declare that they have no conflict of interest.

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Authors and Affiliations

  1. Department of Operative Dentistry and Periodontology, University of Freiburg, Hugstetter Straße 55, 79106, Freiburg, Germany

    Christian Hannig & Bettina Spitzmüller

  2. Clinic of Operative Dentistry, Periodontology and Preventive Dentistry, Saarland University, building 73, 66421, Homburg/Saar, Germany

    Matthias Hannig

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  1. Christian Hannig
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  2. Bettina Spitzmüller
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  3. Matthias Hannig
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Correspondence to Christian Hannig.

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Hannig, C., Spitzmüller, B. & Hannig, M. Characterisation of lysozyme activity in the in situ pellicle using a fluorimetric assay. Clin Oral Invest 13, 15–21 (2009). https://doi.org/10.1007/s00784-008-0213-9

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  • DOI: https://doi.org/10.1007/s00784-008-0213-9

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