THE EFFECT OF HIGH-PRESSURE ON THERMOLYSIN

The effects of high pressure on thermolysin activity and spectroscopic properties were studied. Thermolysin showed distinct pressure-induced activation with a maximum observed at 200-250 MPa for a dipeptide ami de substrate and at 100-120 MPa for a heptapeptide substrate. By exami ning the pressure dependence of the hydrolytic rate for the former sub strate using a high pressure stopped-flow apparatus as a mixing device under elevated pressures, the activation volume of the reaction was - 71 ml mol(-1) at 25 degrees C, Delta V+/- was accompanied by a negativ e activation expansibility and a value of -95 ml mol(-1) was obtained at 45 degrees C. A prolonged incubation of thermolysin under high pres sure, however, caused a time-dependent deactivation. These changes due to pressure were monitored by several spectroscopic methods. The four th-derivative absorbance spectrum showed an irreversible change, mostl y in the tyrosine and tryptophan regions, at a pressure higher than 30 0 MPa. Intrinsic fluorescence and circular dichroism measurements of t hermolysin in solution also detected irreversible changes. All these m easurements indicated that a change occurred at higher pressures and a re explained by a simple two-state transition model accompanied by a l arge, negative change in the volume of reaction.