Statherin is a 43- amino acid residue miniprotein produced by salivary glands. Saliva contains various enzymes which might be able to decompose this peptide. However, not only endogenous enzymes are responsible for a more or less specific turnover of statherin in saliva. Micro-organisms are also potent degradation agents of salivary components.

The purpose of this study was to investigate the stability of statherin in saliva in the presence of a protease inhibitor cocktail and sodium azide.

Samples of whole non-stimulated saliva were collected from four volunteers between 9.00 and 10 a.m. Two hours prior to saliva collection they were refrained from eating, drinking, smoking, and oral hygiene. Saliva from each individual was collected over a 5 min period by spitting into chilled disposable polypropylene tubes. The samples of saliva were mixed and divided into three sub-samples labeled A, B and C. A 1% sodium azide solution was added to sample A in proportion 1 : 1. The protease inhibitor cocktail was added to sample B in proportion 3 : 1. Sample C was a control. The presence of statherin was determined by the matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS) technique. The analyses were performed directly after preparation of the samples and later every 24 hours. The samples of saliva were stored at room temperature (RT). The native statherin has been found to be decomposed in all the samples after 24 hours at RT. Then the synthetic statherin was dissolved in a 10 mM TRIS-HCl buffer of pH 8.2 (concentration 0.5 mg/ml) and was added to samples A, B and C to concentration of 35 μg/ml. In all samples the statherin was decomposed within 48 hours.

Our findings show that the addition of sodium azide, a potent metabolic poison preventing microbial contamination, to whole human saliva did not protect the saliva samples against decomposition of both the native and the synthetic statherin (sample A). A similar situation was noticed in sample B containing the protease inhibitor cocktail . Salivary specific enzymes were inactivated at the beginning of the experiment, but also the native and synthetic statherins were decomposed in that sample.

In this presentation, difficulties encountered in maintaining the stability of the native and synthetic statherins in the three samples will be discussed.

This work was partially supported by the University of Gdańsk (BW 8000-5-0380-7) and the Medical University of Gdańsk ( ST-31) grants.

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