Nitration and Iodination of Human Carbonic Anhydrases: Effects on enzyme activity and on optical properties

Abstract

The chemical modification of the tyrosyl residues in human carbonic anhydrases B and C (EC 4.2.1.1) has been studied in relation to its effects on enzymatic activity, optical rotatory dispersion, and circular dichroism. These studies involved nitration of tyrosyl residues in both enzymes with tetranitromethane and iodination of carbonic anhydrase B. New data on the circular dichroism of the native human carbonic anhydrases, obtained on a JASCO instrument, are reported, and are compared with earlier data of Beychok et al., obtained on a Jouan dichrograph. In general, the data on the two instruments are in close agreement, but some differences are noted, and the new data extend to shorter wave lengths than before. The number of available tyrosyl residues for nitration in carbonic anhydrase B is nearly identical with the number of freely titratable tyrosyls previously estimated by the spectrophotometric titrations of Riddiford. The data reported here show that three tyrosyl groups in carbonic anhydrase B can be nitrated at pH 7 to 8.5; one of these reacts more slowly than the others. Carbonic anhydrase C contains only two tyrosyl groups that can be nitrated under the same conditions. More such groups become available when the reaction occurs at higher pH values, above pH 9 for enzyme C or pH 10 for enzyme B. Nitration of the carbonic anhydrases, at pH 8.5 or below, does not change their enzymatic activities. The optical rotatory dispersion and circular dichroism spectra of the nitrated proteins are indistinguishable from those of the native enzymes, although a new absorption band, at 422 mµ in enzyme B and 428 mµ in enzyme C, is introduced by the formation of 3-nitrotyrosyl residues. Iodination of carbonic anhydrase B caused a decrease in the esterase activity of the enzyme when 2 tyrosyl residues or more were converted to diiodotyrosyl residues. There were accompanying changes in rotatory dispersion and circular dichroism spectra that indicated some loss of the native structure. Urea treatment of the nitrated proteins results in blue shifts of the aromatic absorption bands, similar to the shifts observed for the native enzymes. Although the shapes of the urea difference spectra are very similar for the nitrated and for the native enzymes, slight differences are observed, especially for enzyme B. The urea difference spectra for the nitrated proteins also depend on the urea concentration; the dependence is very similar to that observed for the native enzymes. Urea treatment also exposes the buried tyrosyl groups which become available for nitration. All of the tyrosyls become available for nitration even at urea concentrations at which unfolding of the proteins is not complete as estimated from the difference spectra. Unfolding of the enzymes at high urea concentration is only partially reversible, and refolding depends on the duration of urea treatment. However, Cotton effects in the 250 to 300 mµ region are irreversibly lost in 8 m urea, even in cases in which partial refolding can be shown by difference spectra.