ENVIRONMENTAL SENSITIVITY OF AZO CHROMOPHORES IN ARSANILAZOCARBOXYPEPTIDASE

Coupling of carboxypeptidase A with p-azobenzenearsonate generates a visible, optically active absorption spectrum indicative of arsanilazotyrosine, confirmed by amino acid analysis and measurement of protein-bound arsenic whichfurther reveals the modification of lysine. Physicochemical properties of this derivative are virtually the same as those of the native enzyme. The consequences of modifying O-acetyl-, N-succinyl-, nitro-, and azotetrazolylcarboxypeptidase with p-azobenzenearsonate are consistent with the formation of arsanilazotyrosine which reflects in the visible circular dichroic spectrum. Denaturation of the enzyme with guanidine hydrochloride greatly simplifies the circular dichroic spectrum. The binding of a substrate, glycyl-L-tyrosine, or the inhibitor, β-phenylpropionate, and the removal of the catalytically essential zinc atom markedly alter the complex circular dichroic spectrum in a fashion characteristic for each. The data indicate that the arsanilazotyrosyl chromophore of arsanilazocarboxypeptidase is a sensitive probe of the interactions of β-phenylpropionate or glycyl-L-tyrosine with the enzyme and of the topology of the protein molecule. © 1969, American Chemical Society. All rights reserved.