Liberation of labile sulfur from ferredoxins by alkaline zinc reagent: An appraisal of the methylene blue method

Abstract

In disagreement with reported observation by Suhara and her colleagues ( K. Suhara, S. Takemori, M. Katagiri, K. Wada, H. Kobayashi, and H. Matsubara, 1975, Anal. Biochem. 68, 632–636) we found that more than 90% of labile sulfur was liberated from adrenodoxin within 5 min at 22°C. This rate was faster than those of spinach and clostridial ferredoxins, a result also at variance with Suhara's observation. At low temperature, the reaction was clearly biphasic, and spinach ferredoxin showed a similar profile. In the absence of zinc acetate, activation energies of the decomposition reaction of iron-sulfur center of OH − were obtained as 39, 26, and 11 kcal/mol for adrenal, spinach, and clostridial ferredoxins, respectively. The adrenal reaction became faster as the dipole moment of the solvent increased. In the presence of 4 m urea and 1 m KCl, the rate was enhanced by approximately 26-fold, relative to the reaction without the addition of urea. In conclusion, the liberation reaction of adrenal labile sulfur with alkaline zinc reagent is fast at 22°C, indicating no need for modification of the original method ( T. Kimura and K. Suzuki, 1967, J. Biol. Chem. 242, 485–491; P. E. Brumly, R. W. Miller, and V. Massey, 1965, J. Biol. Chem. 240, 2222–2228).