Conformational stability of bovine holo and apo adrenodoxin — A scanning calorimetric study

Abstract

Holo and apo adrenodoxin were studied by differential scanning calorimetry, absorption spectroscopy, limited proteolysis, and size‐exclusion chromatography. To determine the conformational stability of adrenodoxin, a method was found that prevents the irreversible destruction of the iron‐sulfur center. The approach makes use of a buffer solution that contains sodium sulfide and mercaptoethanol. The thermal transition of adrenodoxin takes place at Ttrs = 46–57 °C, depending on the Na2S concentration with a denaturation enthalpy of ΔH = 300–380 kJ/mol. From ΔH versus Ttrs a heat capacity change was determined as ΔCp — 7.5 ± 1.2 kJ/mol/K. The apo protein is less stable than the holo protein as judged by the lower denaturation enthalpy (ΔH = 93 ± 14 kJ/mol at Ttrs = 37.4 ± 3.3 °C) and the higher proteolytic susceptibility. The importance of the iron‐sulfur cluster for the conformational stability of adrenodoxin and some conditions for refolding of the thermally denatured protein are discussed. Copyright © 2000 The Protein Society