Unfolding and Refolding of Coprinus cinereus Peroxidase at High pH, in Urea, and at High Temperature. Effect of Organic and Ionic Additives on These Processes

The unfolding and refolding rates of the heme- and Ca2+-containing Coprinus cinereus peroxidase (CIP) have been measured at pH 12.1, in 4 M urea, and at 61.2 °C. The change in peroxidase activity paralleled the change in the Soret band absorbance of the heme group. The unfolding rate constant (k u), was determined independently in thermolysin digestion and EDTA experiments at 59.4 °C. Both gave k u values of 1.5 ms-1, and also showed that the presence of 4 mM EDTA made CIP unfolding practically irreversible. Unfolding and refolding rates could therefore be determined under identical conditions of denaturation having either EDTA or Ca2+ in excess. The refolding rates at high pH and in 4 M urea were measured by adding Ca2+ to the unfolded CIP, whereas refolding at 61.2 °C was evaluated by comparing the unfolding carried out under reversible (excess of Ca2+) and irreversible conditions (excess EDTA). The activation energies for the unfolding at 61.2 °C are approximately ΔG ⧧ u 100, TΔS ⧧ u 200, and ΔH ⧧ u 300 kJ/mol. Five different additives, glycerol, EtOH, Na2SO4, guanidinium chloride (GdmCl), and NaCl, all at 100 mM, were used as probes to evaluate the mechanism of base, urea, and heat on unfolding and refolding. Salts destabilized CIP at high pH, primarily by enhancing the unfolding rate but also by decreasing the refolding rate. Glycerol had the reverse effects and thus stabilized CIP at high pH. The unfolding rate in urea was only slightly affected by the additives, with the exception of GdmCl which enhanced the unfolding rate. The refolding rate was decreased in urea by EtOH and GdmCl, in contrast to glycerol and Na2SO4 which increased the refolding rate. At high temperature the unfolding was affected only slightly by the additives, except for GdmCl, and to a lesser extent NaCl, which enhanced the unfolding rate. The refolding rates were greatly decreased by Na2SO4, EtOH, and GdmCl, whereas glycerol and NaCl enhanced the process. It appears that 100 mM NaCl functions as a catalyst for the temperature-induced process, enhancing both the unfolding and refolding rates. The results indicate that the mechanisms of CIP unfolding and refolding are similar in urea and at high temperature but different at high pH.