THERMODYNAMICS OF APOCYTOCHROME-B(5) UNFOLDING

Apocytochrome b5 from rabbit liver was studied by scanning calorimetry , limited proteolysis, circular dichroism, second derivative spectrosc opy, and size exclusion chromatography. The protein is able to undergo a reversible two-state thermal transition. However, transition temper ature, denaturational enthalpy, and heat capacity change are reduced c ompared with the holoprotein. Apocytochrome b5 stability in terms of G ibbs energy change at protein unfolding (DELTAG) amounts to DELTAG = 7 +/- 1 kJ/mol at 25-degrees-C (pH 7.4) compared with DELTAG = 25 kJ/mo l for the holoprotein. Apocytochrome b5 is a compact, nativelike prote in. According to the spectral data, the cooperative structure is mainl y based in the core region formed by residues 1-35 and 79-90. This fin ding is in full agreement with NMR data (Moore, C.D. & Lecomte, J.T.J. , 1993, Biochemistry 32, 199-207).