Effect of pH on the Optical Absorption Spectrum and Effective Number of the Bohr Magneton of Horse Erythrocyte Catalase in the Range from 77°K to 267°K
The optical absorption spectrum of horse erythrocyte catalase [EC 1.11.1.6] in the frozen state was measured at various pH-values at temperatures from 77°K to 267°K. The absorption spectra at pH 8.4 and 11.0, which were predominantly of a high-spin type at room temperature, changed to those of a mixed type of highspin and low-spin forms on cooling to liquid nitrogen temperature. At pH 5.2, on the other hand, the high-spin type character of the spectrum was further intensified on cooling to liquid nitrogen temperature. Magnetic susceptibility measurements were carried out at different pH-values at temperatures from 77°K to 267°K. The square of the effective Bohr magneton number (n2eff) decreased at a given acidic pH, and increased at a given alkaline pH, with the rise in temperature above 180°K. The values of n2eff were temperature independent below 180°K at acidic and alkaline pH-values, being 35, 26.5 and 23.3 at pH 5.0, 9.8 and 10.3, respectively. The EPR absorption spectra of catalase at different pH-values were measured at liquid nitrogen temperature. In the acidic region, the EPR spectrum exhibited a doublet signal with g-values of 6.6 and 5.4 and a weak signal with a Rvalue of 2.03. In the alkaline region, the spectrum exhibited low-spin type signals with values of 2.78, 2.21 and 1.72 in addition to the high-spin type signals described above. These results indicate that (1) the hematin irons of catalase in acidic and alkaline media show more high-spin and more low-spin character, respectively, at low temperatures than at room temperature, and (2) below 180°K the hematin irons in acidic medium are in a pure high-spin state, whereas those in alkaline medium are composed of a mixture of hematin irons in a high-spin state and in a low-spin state. It is considered that there are two forms of hematin in catalase, viz. acidic and alkaline forms, in which an H2 molecule and an OH− ion, respectively, are bound to the sixth coordination position, and that the acidic form, (Fe3+)H2O, is in a purely high-spin state while the alkaline form, (Fe3+)OH−, is in a purely low-spin state at temperatures below 180°K.