Protection of salmon calcitonin breakdown with serine proteases by various ovomucoid species for oral drug delivery.
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abstract
The current work compared protective effects of various ovomucoid species against salmon calcitonin (sCT) metabolism by serine proteases. sCT solutions (50 microM) were incubated at 37 degrees C with trypsin (0.5 microM), alpha-chymotrypsin (0.1 microM), or elastase (0.48 microM) in 50 mM Tris buffer (pH 8.0) containing or lacking different concentrations of turkey ovomucoid (tOVM), duck ovomucoid (dOVM), or chicken ovomucoid (cOVM) and aprotinin. Caco-2 cell homogenate was also incubated with sCT and the contents of the proteases were assayed by using their specific substrates. Metabolites were identified by high-performance liquid chromatography, gel electrophoresis, and matrix-assisted laser desorption ionization mass spectrometry techniques. In the absence of inhibitors, there was a considerable degradation of sCT by the proteases. dOVM and tOVM increased the half-life of sCT with trypsin and alpha-chymotrypsin at enzyme-to-inhibitor ratio of 1:4 showing similar efficacy. dOVM was found to be superior to tOVM in protecting sCT from elastase. cOVM was ineffective in protecting sCT against alpha-chymotrypsin. Caco-2 cell homogenate degraded sCT, which was prevented by tOVM. sCT was cleaved into different molecular weight fragments with different proteases. In general, the metabolite formation decreased when inhibitor concentration increased. dOVM and tOVM effectively stabilized sCT against all three proteases. However, cOVM could not prevent the degradation by alpha-chymotrypsin.
