Effects of denaturing agents on the phenylalanyl circular dichroism bands of horseradish peroxidase isoenzymes and apoisoenzymes.

The influence of protein conformation upon the circular dichroism (CD) bands of two peroxidase isoenzymes (Al and C) was investigated by using guanidine hydrochloride and other denaturing agents. In all four proteins, guanidine hydrochloride markedly reduces the Zl-nm CD band, indicating disruption of the ordered peptide backbone conformation. Similarly guanidine hydrochloride greatly diminishes the near ultraviolet CD bands of the tyrosyl and tryptophanyl side chains, which implies that their orientation has been disrupted. In contrast, guanidine hydrochloride treatment of peroxidase C brings out phenylalanyl CD fine structure at 268 and 261 nm, even though none can be detected in the native conformation. The intensities of these CD bands in the denatured peroxidase C are comparable to those of an equivalent concentration of unoriented phenylalanyl model compound. These findings indicate that in the native peroxidase C many of the phenylalanyl side chains have fixed orientations producing unique interactions with the surroundings. Apparently the rotatory strength of each oriented phenylalanyl side chain may be weak or strong and either positive or negative. The native peroxidase C may have as many as 23 oriented phenylalanine residues; summing the individual contributions leads to nearly complete cancellation of CD intensity. In the denatured peroxidase C, even though the unoriented phenylalanyl side chains have weak CD bands, all have nearly the same CD spectrum, thereby precluding cancellation among the residues. Thus the observed intensity of the phenylalanyl CD bands increases when the orientations of the phenylalanyl side chains are disrupted in peroxidase C.