Parotid saliva placed in 35-mm-diameter tissue culture dishes developed increasing surface viscoelasticity at the interface with air. A surface layer became visible with time, and was collected and analysed by protein electrophoresis which indicated that a single protein (pI 4.2; molecular mass approx. 6 kDa) predominated. Western blot analysis demonstrated that the major protein band reacted with an antiserum directed against the C-terminal of the calcium-binding salivary protein statherin. Matrix-assisted laser-desorption ionization-time-of-flight MS analysis gave a molecular mass of 5380 Da for the protein, corresponding to the molecular mass of statherin. Staining of film protein in electrophoresis gels was compared with statherin synthesized on a solid phase, and the mean statherin content of film formed from 1 ml of parotid saliva was measured as 7 nmol. The mean calcium content of the surface layer was 250 nmol. Surface rheology was greatly decreased in the presence of EDTA, whereas surface tension of saliva was unaffected by calcium chelation, suggesting that protein accumulated at the surface was unaffected. The results suggest that a layer rich in statherin forms at the interface of saliva and air, and that the surface rheology developed is dependent upon protein interactions mediated by calcium. The surface layer may enhance the function of saliva as a protective layer on the mucosal surfaces and teeth.
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