Conformational stability of bovine holo and apo adrenodoxin--a scanning calorimetric study.
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.
Burova
T V
TV
Institute of Food Substances, Russian Academy of Sciences, Moscow.
Bernhardt
R
R
Pfeil
W
W
D000325:Adrenodoxin / Q000737:chemistry* / Q000378:metabolism; D000818:Animals; D001059:Apoproteins / Q000737:chemistry / Q000378:metabolism; D001205:Ascorbic Acid / Q000494:pharmacology; D002152:Calorimetry, Differential Scanning; D002417:Cattle; D002918:Chymotrypsin / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D008623:Mercaptoethanol / Q000494:pharmacology; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D013053:Spectrophotometry; D013440:Sulfides / Q000494:pharmacology; D013696:Temperature; D013816:Thermodynamics; D014508:Urea / Q000494:pharmacology
D001059:Apoproteins; D013440:Sulfides; D000325:Adrenodoxin; D008623:Mercaptoethanol; D014508:Urea; D002918:Chymotrypsin; D001205:Ascorbic Acid; C033479:sodium sulfide