Further studies of lobster carbonic anhydrase

Abstract

Proteins extracted from lobster hemocytes by differential detergent fractionation (Ramsby & Makowski, 1999) were analyzed for carbonic anhydrase (CA) activity by hydration of CO2 in bromthymol blue-stained gels. We have adopted the method of Bundy (1986) to remove SDS from PAGE gels (incubation with the ion-retardation resin BioRad AG11A8) in order to estimate polypeptide sizes and to correlate those data with the labeling pattern obtained with anti-Human CA I on a Western blot of the same gel. While the Wilbur reaction is difficult to quantitate, the yellow spots are diffuse and fade, their positions can be marked by holes that persist through the electroblotting and immunodetection steps. What we observe is intense labeling of a ca. 40 kDa band, but also bands at ca. 75 kDa and 200 kDa. A curious observation with the lobster CA is the persistent association of CA activity with the 75 kDa hemocyanin subunit when the hemocyte supernatant is analyzed and in the first DDF extract (possible contamination by the very high concentration of hemocyanin). It is interesting that Bundy (1986) found the C. reindardii CA to be 42 kDa. *Supported by the Altman Fund.