The location of tryptophanyl groups in human and bovine carbonic anhydrases: Ultraviolet difference spectra and chemical modification

Abstract

1. 1. The effects of 20% sucrose, glycerol, ethylene glycol, polyethylene glycol, dimethyl sulfoxide and 86% 2H 2O on the ultraviolet absorption spectra of bovine carbonic anhydrase and the human carbonic anhydrases B and C have been investigated. The results suggest that 3 ± 1 out of 7 tryptophan residues are accesible to the solvent in the bovine and human C enzymes and 2 ± 0.5 residues out of 6 in the human B enzyme. There is no systematic dependence of the degree of accessibility on the size of the perturbing molecule, but “short range” perturbants consistently give lower values than “long range” perturbants. 2. 2. In the bovine enzyme and the human C enzyme no tryptophan residue appears to have a “normal” reactivity toward 2-hydroxy-5-nitrobenzyl bromide. In the human B enzyme the equivalent of one residue can be modified without significant losses of enzymic activity and native structure. 3. 3. Acid denaturation appears to result in an incomplete exposure of tryptophanyl groups. Addition of urea or guanidine·HCl to the acid-denatured enzyme results in spectral changes suggesting a further unfolding of the molecule. It is estimated that the transfer of all the tryptophanyl groups of the native bovine enzyme to water is associated with Δε 292 nm approximately — 11 000 M −1 · cm −1 or an average value per tryptophan residue approximately −1600 M −1 · cm −1. 4. 4. A survey of a model of the crystal structure of human carbonic anhydrase C has been made. Only one of the tryptophan residues is buried in the interior of the molecule. The remaining 6 groups have surface positions. One of these surface groups would be considerably more exposed to the solvent than the remaining 5 groups.