Elasmobranch Carbonic Anhydrase: PURIFICATION AND PROPERTIES OF THE ENZYME FROM TWO SPECIES OF SHARK

Abstract

Homogeneous carbonic anhydrase has been prepared from erythrocytes of the bull and tiger shark. Molecular and equivalence weight determinations indicate molecular weights in the range of 36,000 to 40,000 depending on the method employed. Amino acid compositions are similar to those reported for mammalian carbonic anhydrases with several notable exceptions. The bull and tiger shark enzymes contain ∼25 and 18 half-cystine residues per molecule, respectively. These residues are apparently present as disulfide cross-links, since neither the native nor denatured molecules react with sulfhydryl reagents, but do react after reduction with mercaptoethanol. There are high contents of glutamic and aspartic acid compatible with the very acidic nature of these proteins. The isoelectric point of the bull shark enzyme is ∼pH 4.5. The shark enzymes catalyze both the hydration of CO2 and the hydrolysis of certain aromatic esters. Maximal catalytic rates are comparable to the high activity forms of mammalian carbonic anhydrases. Aromatic sulfonamides are powerful inhibitors. Circular dichroic spectra in the ultraviolet region indicate the presence of many structural features similar to those found in the mammalian enzymes. The shark enzymes contain 1 g atom of zinc per mole of enzyme. A stable metal-free, inactive apoenzyme can be prepared which is completely reactivated by Zn(II) or Co(II) ions. The Co(II) derivatives have intense visible absorption maxima at 510, 555, 618, and 645 nm. These spectra, as well as the spectra of sulfonamide complexes of the Co(II) enzymes, are almost identical with those of the Co(II) derivatives of mammalian carbonic anhydrases, and suggest that similar coordination geometries exist at the active centers of the elasmobranch and mammalian enzymes. Since fossil morphological evidence suggests that present day shark species have not evolved much since the Devonian period, this particular type of metal complex would appear to have been a constant feature of carbonic anhydrase over 400 million years of animal evolution.