Quantitative electron spin resonance studies on native and denatured ceruloplasmin and laccase

Abstract

The effect of urea denaturation on the visible absorption, electron spin resonance spectrum and oxidase activity of ceruloplasmin has been investigated. Some measurements of the effect on optical rotation and ultraviolet absorption have also been performed. The denaturation destroys the specific bonding of Cu 2+ , characteristic for native ceruloplasmin as evidenced by electron spin resonance spectra. The loss of color and oxidase activity follows the same time-course and concentration dependence as the change in bonding, while comparable changes in optical rotation and ultraviolet difference spectra require more drastic denaturation. These findings indicate that the strong visible absorption is related to the specific bonding of Cu 2+ , and that this bonding is necessary for catalytic activity. With both ceruloplasmin and laccase, the Cu 2+ signal of the native protein only corresponds to about 50% of the total copper content, as determined both by chemical analysis and by measurements of the electron spin resonance signal intensities after release and oxidation of protein-bound copper with perchloric acid. The possibility that the specific bonding may involve an interaction between Cu 2+ and Cu 1+ is discussed.