Carbonic anhydrase in mineralization of the crayfish cuticle

The cuticle of the freshwater crayfish, Procambarus clarkii (Girard, 1852), has several extracellular, cuticular isozymes for carbonic anhydrase (CA) which were isolated and purified via affinity chromatography. To denature proteins not entombed in cuticle calcite the ground cuticle was washed twice with both a 0.5% SDS and 1.5% sodium hypochlorite solution prior to CA isolation. Total water soluble protein (WSP) was 133 +/- 68 (SD) mg/100 g (6) cuticle, whereas affinity column bound protein, CA, was 191 +/- 108 (SD) mug/100 g (6) cuticle. CA was 0.144% WSP. EDTA dissolved the calcium carbonate and freed the CA along with the other WSP. The CA fraction was positive to bovine CA II antibodies (ELIZA and Western Blot) and had low CA enzyme activity. The estimated molecular weights using PAGE showed one isozyme with a molecular weight of 29 kDa, which is similar to that in vertebrates, and two larger isozymes (53 and 66 kDa). All four isozymes were detected in only one sample, whereas the two larger isozymes were detected in all six cleaned cuticle samples. The larger isozymes may be dimers of the smaller isozyme and are more like those detected in oyster pearls and shell with weights of 60 kDa.