Carbonic Anhydrase-Azosulfonamide Complexes: SPECTRAL PROPERTIES

Abstract

Abstract Carbonic anhydrase combines with the optically inactive azosulfonamide (disodium 2-(4-sulfamylphenylazo)-7-acetamido-1-hydroxynaphthalene-3,6-disulfonate) to form a highly colored enzyme-inhibitor complex. Large visible Cotton effects ([M] = 120,000°) are induced in the multiple visible absorption bands (e500 = 25,000) of the phenylazonaphthol chromophore by the dissymmetric environment of the protein binding site. Bathochromic-hypochromic shifts of these bands accompanying binding indicate the presence of a hydrophobic binding site. Circular dichroism spectra of azosulfonamide complexes of five isozyme and species variants of carbonic anhydrase reveal that both sign and magnitude of the multiple large visible ellipticity bands ([θ] = ± 0.5 - 8.0 x 104 deg cm2 per decimole, Rk = ± 0.4 - 19.7 x 10-39 c.g.s.u.) are unique for each isozyme. Thus the azosulfonamides become sensitive probes for conformational features of the active center of carbonic anhydrase. Ultraviolet ellipticity bands ([θ] = 1.5 x 106 deg cm2 per decimole) induced in the phenylazonaphthol chromophore are large enough in certain isozyme complexes to completely dominate both the circular dichroism and the rotatory dispersion of the enzyme-inhibitor complexes. In accordance with the metal ion specificity observed for the induction of hydration or esterase activity in apocarbonic anhydrase, only Zn(II) and Co(II) at the active site induce the specific binding of the azosulfonamide which imparts optical activity to the visible absorption bands of the sulfonamide. Marked differences in the pattern of ellipticity bands induced in the sulfonamide by the Zn(II) and Co(II) enzymes, however, indicate participation of the metal ion in determining the final conformation of the bound sulfonamide or the active center as well as determining the binding affinity of the enzyme for the sulfonamide inhibitors.