Biocalcification and Activity of Carbonic Anhydrase in the Extracellular Matrix of Coral

Abstract

Crushed exoskeletons of the coral, Astrangia astrangia, were treated with sodium hypochlorite (1.5%), SDS (0.5%) and then decalcified with EDTA. Upon purification by affinity chromatography extracellular proteins with Mr of 31, 53, and 63 kDa were distinguished using SDS/PAGE gel. The proteins appear to be extracellular isozymes of carbonic anhydrase (CA). Bovine serum CAII antibodies cross-reacted with the proteins isolated via affinity chromatography (ELISA and Western Blots). High CA enzyme activity and the inhibition of CA with acetazolamide, all support the contention that an extracellular CA is present in the exoskeleton. Cleaning the crushed exoskeleton with both SDS and sodium hypochlorite to remove cellular proteins that are not entombed in the calcite prior to CA extraction we assert that CA is secreted extracellularly along with the matrix proteins. The total water-soluble proteins (WSP) in the exoskeleton of the coral, A. astrangia, was 14 + 7 mg - protein/lOOg exoskeleton whereas CA isozymes represent 224 ± 108 |Xg - protein/lOOg exoskeleton (N=6) or 1.71% the total WSP. These data illustrate that coral extracellular CA isozymes do function in exoskeleton mineralization by maintaining the bicarbonate concentrations at equilibrium so as to maintain the availability of carbonate ions used in mineralization.