PURIFICATION AND CHARACTERIZATION OF A CARBONIC ANHYDRASE-II INHIBITOR FROM PORCINE PLASMA

Plasma from many vertebrates, including pigs, contains a soluble component that inhibits the CO2 hydrase activity of carbonic anhydrase (CA). This activity was purified to homogeneity (approximately 4000-fold) from porcine plasma using a combination of DEAE-Affi-Gel Blue chromatography and carbonic anhydrase II-affinity chromatography, yielding 16 mg of inhibitory protein/L of plasma. This protein, porcine inhibitor of carbonic anhydrase (pICA), is a monomeric protein with an apparent molecular mass of 79 kDa, as determined by electrospray mass spectrometry. As isolated, pICA contains about 3 kDa of N-linked glycosylation removable by peptide-N-glycosidase F. pICA inhibits CA reversibly with a 1:1 stoichiometry. pICA is a potent and specific inhibitor of the CA II isozyme, with K(i) < 0.1 nM for porcine CA II at pH 7.4. Although the K(i) is dependent on the CA isozyme type (CA II << CA IV << CA III almost-equal-to CA I), it is relatively insensitive to the species source, as long as it is mammalian. The K(i) is pH dependent with log K(i) decreasing linearly as the pH decreases, implicating at least one ionizable group with the pK(a) less-than-or-equal-to 6.5 in the binding interaction. The isozyme and species dependence of the inhibition suggest that pICA interacts with amino acids on the surface of CA II.