Peroxidase Isoenzymes from Horseradish Roots: III. Circular Dichroism of Isoenzymes and Apoisoenzymes

Abstract

Abstract The conformations of two peroxidase isoenzymes (A1 and C) and their apoenzymes were investigated by means of circular dichroism. In the visible region the circular dichroism of the two isoenzymes is essentially identical in five of the seven optically active heme absorption bands. These results suggest that the active sites are similar in both isoenzymes, but that small differences do exist. In the ultraviolet region both isoenzymes have the same circular dichroism (CD) with a positive band at 191 mµ and with negative bands at 208 and 221 mµ, suggesting that the isoenzymes have an appreciable helical content. When the heme is removed from either isoenzyme, the intensities of these bands are reduced by 14 to 28%. In the 250 to 290 mµ range, both isoenzymes have a negative CD band at 286 mµ and fine structure CD bands at 262 and 268 mµ. In addition, C peroxidase has a band at 280 mµ, and A1 peroxidase has a shoulder at 291 mµ. After removal of the heme from the isoenzymes, the 286 mµ CD band is lost, and the differences between the CD spectra of the two apoperoxidases are clearly evident. The C apoperoxidase has a negative CD band at 278 mµ. The A1 peroxidase has its main band at 283 mµ and a shoulder at 291 mµ. Both apoperoxidases retain the fine structure bands at 262 and 268 mµ. From a comparison of the peroxidase absorption spectra and the positions of CD bands in model compounds, the chromophores of the peroxidase CD bands are tentatively identified as: 286 mµ, heme; 278 mµ, tyrosyl; 283 and 291 mµ, tryptophanyl; 262 and 268 mµ, phenylalanyl.