On the Acid Denaturation of Porcine Erythrocyte Catalase in Relation to Its Subunit Structure1

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Abstract
Porcine erythrocyte catalase [EC 1.11.1.6] (molecular weight, ca. 250, 000) was found to dissociate partially between pH 3.5 and 3.0 and completely below pH 3.0 into two presumably identical 1/2-sized subunits (molecular weight, 112, 000) as estimated by ultracentrifugal analyses. This dissociation was accompanied by a marked change in hydrodynamic properties; the sedimentation coefficient decreased from about 11S to 4S. This acid denaturation also resulted in complete loss of enzyme activity and disappearance of absorption bands characteristic of heme protein, in particular, a shift of the Soret band from 405 nm to a small and broad band at 375 nm. The change in enzyme activity correlated well with that of the Soret band, depending on the denaturation time and pH used. Reversible recovery of enzyme activity was not detected below pH 3.1 after 2 h denaturation. The pH dependence of α-helical content estimated from the CD intensity at 222 nm also correlated well with that of enzyme activity. The rate constants of initial reaction of acid denaturation at several pHs were determined by following the changes in the Soret band with time, since the changes showed an isosbestic point at 384 nm. The results revealed that the first-order rate constant at pH 3.0 was 45 times larger than that at pH 3.4, indicating that the rate of acid denaturation increased rapidly within a narrow acidic pH range. The temperature dependence of the denaturation rate was also measured and the activation energy for the acid denaturation was found to be 76.3 kcal/mol from an Arrhenius plot.

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