Spectrophotometric analysis of the interaction between cytochrome b5 and cytochrome c

Abstract

The interaction between [horse heart] cytochrome c and the tryptic fragment of [beef liver] cytochrome b5 produces a difference spectrum in the Soret region with a maximum absorbance at 416 nm. The intensity of this difference was used to determine the stoichiometry of complex formation and the stability of the complex formed. At pH 7.0 [25.degree. C (phosphate), .mu. [ionic strength] = 0.01 M], the 2 proteins formed a 1:1 complex with an association constant, KA, of 8 (3) .times. 104 M-1. The stability of the complex was strongly dependent on ionic strength with KA increasing to 4 (3) .times. 106 M-1 at .mu. = 0.001 M [25.degree. C, pH 7.0 (phosphate)]. Analysis of the dependence of KA on pH from pH 6.5-8 demonstrated that this complex is maximally stable between pH 7 and 8 or about midway between the isoelectric points of the 2 proteins. Analysis of the temperature dependence of KA revealed that formation of the complex between the 2 proteins is largely entropic in origin with .DELTA.H.degree. = 1 .+-. 3 kcal/mol and .DELTA.S.degree. = 33 .+-. 11 eu [entropy units] [pH 7.0 (phosphate), .mu. = 0.001 M]. This result may be explained either by the model of Clothia and Janin in terms of extensive solvent reorganization upon complexation or by the model of Ross and Subramanian in which the negative enthalpic and entropic contributions of short-range protein-protein interactions are offset by proton release.