Denaturation of Aplysia myoglobin. Equilibrium study

Abstract

The effect of temperature and solvent composition on the reversible denaturation of Aplysia myoglobin is reported here. The denaturation is accompanied by changes in the haem absorption spectrum, in the intrinsic fluorescence of the protein and in its optical activity in the Soret and ultraviolet regions. Under all conditions examined (i.e. different temperatures, different concentrations of various alcohols and different concentrations of proteins) the system appears to be in a state of true equilibrium and good agreement is found between thermally-induced and solvent-induced transitions. The equilibrium behaviour is consistent with a simple all-or-none model. The same transition is observed when different physical parameters are monitored. The effect of the various alcohols appears to be essentially entropic, since the van't Hoff ΔH∘ is nearly independent of the relative concentration of ethanol (from 0 to ~25% v v ). Moreover, within the errors of the measurements, the van't Hoff heat is independent of temperature, indicating that the Δ Cp cannot be very large. The destabilizing effect of alcohols, dealt with in the framework of a minimum scheme, is considered in terms of differential interaction of the third component with the two forms of the protein. A minimum estimate of the number of alcohol molecules differentially bound by the native and denatured conformations, based on linkage treatment, is found to be nearly independent of the type of alcohol used.