COLD DENATURATION AND HEAT DENATURATION OF STREPTOMYCES SUBTILISIN INHIBITOR .1. CD AND DSC STUDIES

Cold denaturation and heat denaturation of the protein Streptomyces subtilisin inhibitor (SSI) were studied in the pH range 1.84-3.21 and in the temperature range -3-70-degrees-C by circular dichroism and scanning microcalorimetry. The native structure of the protein was apparently most stabilized at about 20-degrees-C and was denatured upon heating and cooling from this temperature. Each denaturation was reversible and cooperative, proceeding in two-state transitions, that is, from the native state to the cold-denatured state or from the native state to the heat-denatured state. The two denatured states, however, were not perfect random-coiled structures, and they differed from each other, indicating that there exist three states in this temperature range, i.e., cold denatured, native, and heat denatured. The difference between the cold and heat denaturations was indicated first by circular dichroism. The isodichroic point for the transition from the native state to the cold-denatured state was different from that from the native state to the heat-denatured state in the pH range between 3.21 and 2.45. Moreover, molar ellipticity for the cold-denatured state was different from that of the heat-denatured state, and the transition from the former to the latter was observed at pH values below 2. Values of van't Hoff enthalpies from the native state to the heat-denatured state at pH values between 3.21 and 2.45 were obtained by curve fitting of the CD data, and DELTA-C(p) = 1.82 (+/- 0.11) [kcal/(mol.K)] was obtained from the linear plot of the enthalpies against temperature. The parameters obtained from the heat denaturation studies gave curves for DELTA-G-degrees which were not in agreement with the experimental data in the cold denaturation region when extrapolated to the low temperature. Moreover, the value of the apparent DELTA-C(p) the cold denaturation in the pH range 3.03-2.45 was estimated to be different from that for the heat denaturation, indicating that the mechanism of the cold denaturation of SSI is different from a simple cold denaturation. A differential scanning calorimetric study confirmed that the heat denaturation was of the cooperative two-state type (from the dimeric form in the native state to the monomeric form in the heat-denatured state) with absorption of enthalpy upon increasing the temperature at pH 3.07. As the pH decreased, however, the enthalpy for the heat denaturation decreased whereas that for the cold denaturation showed a different behavior. The value of the heat capacity of the protein in the cold-denatured state is different from that in the heat-denatured state and is even closer to that in the native state. Furthermore, below pH 2, a direct transition between the two denatured states was observed with absorption of heat.