- Carbonic anhydrase (CA) was purified from erythrocytes of rainbow trout (Salmo gairdneri) and biochemically characterized. Purification involved hemoglobin denaturation by chloroform-ethanol treatment, Sephadex G-75 gel filtration, and DEAE Biogel A anion exchange chromatography. Protein homogeneity was verified by polyacrylamide gel electrophoresis under native and denaturing conditions and by polyacrylamide gel isoelectric focusing., A single enzyme form was found in trout erythrocytes. The enxyme's high CO(,2) hydration specific activity, amino acid composition, and sensitivity to sulfonamide inhibition all suggest that it is a CA II isozyme. Its molecular weight was 28,000 as determined by sodium dodecyl sulfate gel eclectrophoresis, and pI was 9.3. The enzyme contained one mole of zinc per mole of CA. Trout CA was also active as an esterase, with K(,m) = 7.3 mM and k(,cat) = 175 min('-1) at pH 7.5 when p-nitrophenyl acetate was the substrate., Amino acid analysis indicated the presence of six half-cystine residues per molecule of trout CA, and maintenance of a reducing environment was required for optimal activity. Removal of reducing agent by dialysis resulted in about 80% loss of activity; this activity loss was reversible. Sephadex G-75 chromatography in the absence of a reducing agent demonstrated the presence of low-activity CA dimers, but dimer formation was not a prerequisite for inactivation, as remaining CA monomers were likewise inactivated., and Sulfhydryl modification studies using both N-ethylmaleimide and acrylonitrile indicated that three reactive sulfhydryl groups were present in the reduced enzyme. Modification of these groups had no effect on enzyme activity, but modified CA was no longer subject to inactivation by oxidizing conditions.
- Dissertation Note:
- Ph.D. The Pennsylvania State University 1982.
- Source: Dissertation Abstracts International, Volume: 43-07, Section: B, page: 2187.
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