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Biochimica et biophysica acta

Homotropic cooperative binding of organic solvent vapors by solid trypsin.


PMID 11342057

Abstract

Homotropic cooperative binding was observed at vapor sorption of organic solvents (acetonitrile, propionitrile, ethanol, 1-propanol, 2-propanol, nitroethane) by dried solid trypsin from porcine pancreas (0.05 g H2O/g protein). The vapor sorption isotherms were obtained by the static method of gas chromatographic headspace analysis at 298 K for 'vapor solvent+solid trypsin' systems in the absence of the liquid phase. All isotherms have a sigmoidal shape with significant sorbate uptake only above the threshold of sorbate thermodynamic activity. On the sorption isotherms of non-hydroxylic sorbates the saturation of trypsin by organic solvent was observed above the sorbate threshold activity. The formation of inclusion compounds with phase transition between solvent-free and solvent-saturated trypsin is supposed. Approximation of obtained isotherms by the Hill equation gives the inclusion stoichiometry S, inclusion free energy, and the Hill constant N of clathrates. The inclusion stoichiometry S depends significantly on the size and shape of sorbate molecules and changes from S=31 mol of sorbate per mol of trypsin for ethanol to S=6 for nitroethane. The inclusion free energies determined for the standard states of pure liquid sorbate and infinitely dilute solution in toluene are in the range from -0.5 to -1.2 kJ/mol and from -3.1 to -8.1 kJ/mol, respectively, per 1 mol of sorbate. The Hill constants are relatively high: from N=5.6 for 1-propanol to N approximately equal to 10(3) for nitroethane. The implication of the obtained results for the interpretation of solvent effects on the enzyme activity and stability in low-water medium is discussed.

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