alpha-chymotrypsin and sodium-sulfide

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 degrees C, depending on the Na2S concentration with a denaturation enthalpy of delta H = 300-380 kJ/mol. From delta H versus Ttrs a heat capacity change was determined as delta Cp = 7.5 +/- 1.2 kJ/mol/K. The apo protein is less stable than the holo protein as judged by the lower denaturation enthalpy (delta H = 93 +/- 14 kJ/mol at Ttrs = 37.4 +/- 3.3 degrees 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.

Topics: Adrenodoxin; Animals; Apoproteins; Ascorbic Acid; Calorimetry, Differential Scanning; Cattle; Chymotrypsin; Electrophoresis, Polyacrylamide Gel; Hydrogen-Ion Concentration; Mercaptoethanol; Protein Conformation; Protein Denaturation; Protein Folding; Spectrophotometry; Sulfides; Temperature; Thermodynamics; Urea