Kinetics of transfer in aqueous solution of ferriprotoporphyrin IX from human serum albumin to sperm whale apomyoglobin

Abstract

SynopsisKinetic aspects of the transfer reaction of ferriprotoporphyrin IX (FP) from its complex with human serum albumin (HSA) to sperm whale apomyoglobin were investigated by spectrophotometry in aqueous solution. At molar ratios of 2 1 of both HSA and apomyoglobin to FP (1 X 10-5M), the initial rate of transfer at pH 7.4 (0.025M Tris buffer) and 25'C was virtually independent of the concentration of apomyoglobin up to a sixfold excess of the latter. The rate-limiting step in the transfer reaction is considered to be the dissociation of FP from the FP-HSA complex with an apparent rate constant of the order of s-l under the above conditions. The initial rate decreased with increasing concentrations of HSA, indicating competition between HSA and apomyoglobin for free FP in the recombination reactions. A steady-state concentration of the order of 10-9M is estimated for free FP under the conditions given. The initial rate of transfer decreased markedly with pH in the range of pH 5-9 and was also dependent on the type and concentration of the buffer used. Also, various electrolytes at different concentrations showed very different and specific effects on the initial rate of reaction. Similarly, various drugs and analogous substances such as penicillins and salicylate affected the rates of dissociation in different concentration ranges. From the temperature dependence of the initial rate at pH 7.4 in the range of 5-25"C, an overall energy of activation of about 8 kcal/mol and an entropy of activation of about -50 e.u. are evaluated, indicating a much higher order of molecular groups around FP or other steric constraints in the transition state. It appears that kinetic parameters of the hiopolymer-small molecule system are more sensitive to environmental changes than corresponding equilibrium data as measured by light absorption.

Probing the Differences between Rat Liver Outer Mitochondrial Membrane Cytochrome b5 and Microsomal Cytochromes b5

“…∆A eq values at equilibrium were calculated using 406 ) 151 mM -1 cm -1 for holoMb and 406 ) 122 mM -1 cm -1 for holocyt b 5 . These values are based on Soret band λ max values reported in the literature for Mb (45) and cyt b 5 (23).…”