Studies on the Peroxidase Effect of Cytochrome c. III. A Kinetic Study of the Over-all Reaction.

Abstract

The peroxidation of pyrogallol to purpurogallin, catalyzed by ferricytochrome c, satisfied the criteria of an enzymic reaction, which fitted the properties of a "true" peroxidase; i.e. the catalytic activity exhibited a definite pH "optimum" of about 3.5 (40 mM citrate buffer; 25[degree]C), and was a linear function of the concentration of the hemoprotein. The apparent optimum concentration of either reactant (H2O2 and pyrogallol) was found to be higher, the higher the concentration of the other; no definite optimum concentrations can therefore be given. In a double reciprocal plot, the "initial" reaction rate was proportional to the concentration of pyrogallol and to the square of the concentration of H2O2. A high concentration of this reactant was always required to obtain a measurable reaction rate. Cytochrome c was progressively destroyed during the reaction, and this destruction proceeded more rapidly, the higher the concentration of H2O2 and the temperature. The reaction was further characterized by a nonlinear Arrhenius plot and a high apparent activation energy (E'' 22,200 cal mole-1) within the temperature range 15-25[degree]C. Ferrocytochrome c was just as effective as the ferric form, inasmuch as it is rapidly oxidized to the trivalent condition. The peroxidase reaction is a sensitive and accurate method for the assay of cytochrome c. In the standard procedure of the present study, it was calculated to be approximately 40 and 130 times more sensitive than the spectrophotometric determination of the absorbancy in the [alpha]-band of ferrocytochrome c when the reaction product was measured after 1 and 10 min, respectively. The sensitivity could, however, be further increased by the use of higher concentration of the substrates and/or of buffer anions.