Calorimetric and High Pressure Fluorescence Studies of the Salt Effects on Thermolysin

The relationship of the salt-activation of thermolysin and the thermodynamic properties was investigated by using DSC and high-pressure spectrofluorometry. As previously reported [Holmquist and Vallee, Biochemistry, 15, 101 (1976), Fukuda and Kunugi, Biocatalysis, 2, 225 (1989)], 1:1 salts such as Na+ and K+ with Cl− or Br− showed monotonous increase in the activity, and the rankings of rate increase at 2 M were Na+ ≍ K+ > Ca2+ > Mg2+ and Cl− ≍ Br− > I− ≍ SO42− >SCN−. In the presence of salt, thermolysin showed lower peak temperature in DSC, and the instability was in the order of SCN− > I− > Br− > Cl− and Ca2+ > Mg2+ > Na+ > K+. By addition of 0.5 M NaBr, the contour map of the peak intensity of thermolysin intrinsic fluorescence became much simpler, and the contour shifted to lower temperature. The −ΔV values of the transition decreased with increasing salt at lower concentration range, but then increased at higher concentration, especially at higher temperature. Thus ΔV became more negative with increasing temperature. These results are discussed with respect to the ion distribution among the low- and high-density water phases in the protein solution, and the surface charge, hydration, and flexibility of enzyme protein, especially at the transition state of the catalysis.