Purification and Properties of Horse Erythrocyte Carbonic Anhydrases

A J Furth

doi:10.1016/s0021-9258(18)93194-7

Abstract

Abstract Two distinct components of carbonic anhydrase (EC 4.2.1.1) have been purified from horse red cells. In the isolation procedure described, the large excess of hemoglobin was removed by precipitation in ammonium sulfate, and the two carbonic anhydrase proteins were then separated by DEAE-Sephadex chromatography. This separation was facilitated by the remarkably high isoelectric point of the basic component, which appeared to have a net positive charge even at pH 10.0. This basic component, which was designated enzyme C, constituted only 36% of the total carbonic anhydrase protein but had a high specific activity; with the use of the WilburAnderson assay for CO2 hydration, this was in the order of 40,000 units per mg. The major protein, designated enzyme B, had a much lower specific activity, in the order of 4,000 units per mg. The extinction coefficients and s20,w values of the purified B and C proteins were measured, as well as the Km and Vmax values for their hydrolysis of p-nitrophenyl acetate. The amino acid compositions and the optical rotatory dispersion spectra are also reported. The two components of horse carbonic anhydrase described here give a further example of the unusual type of polymorphism already seen with primate carbonic anhydrase. This suggests that it may be a characteristic of the enzyme from the blood of a variety of mammalian species. The salient features seem to be firstly, the presence of two chemically distinct proteins catalyzing the same reaction at different rates, and secondly, the much greater abundance of the less active form. The isolation of two horse enzymes brings to seven the number of carbonic anhydrases now studied in a highly purified state. These are classified as either high specific activity forms (human, rhesus monkey, and horse C enzymes, bovine B enzyme) or low specific activity forms (human, rhesus monkey, and horse B enzymes), and the proteins in each category are then compared in terms of amino acid composition, kinetics, and optical rotatory dispersion.

Highlights

4.2.1.1) have been purified from horse red cells
In the isolation procedure described, the large excess of hemoglobin was removed by precipitation in ammonium sulfate, and the two carbonic anhydrase proteins were separated by DEAE-Sephadex chromatography
This separation was facilitated by the remarkably high isoelectric point of the basic component, which appeared to have a net positive charge even at pH 10.0. This basic component, which was designated enzyme C, constituted only 36% of the total carbonic anhydrase protein but had a high specific activity; with the use of the Wilbur-Anderson assay for CO2 hydration, this was in the order of

Summary

Methods

Method BDenaturation with ethanol and chloroform was used. This method differed only slightly from the procedure described in Reference 14. The proportions used were 700 ml of hemolysate to 125 ml of ethanol and 150 ml of chloroform, and t,he procedure was modified by the addition of 50 ml of 1 M phosphate buffer at pH 6.8 immediately before the addition of chloroform This raised the ionic strength and reduced the amount of carbonic anhydrase lost by adsorption to denatured hemoglobin. The large volumes of dilute protein solution resulting from each of the above procedures could be rapidly reduced by use of solid ammonium sulfat,e, if the pH were first adjusted to 7 with concent,rated phosphate buffer. This was part.icularly important for solutions previously at high pH to prevent the release of free ammonia. Protein inside the sacs precipitated in the resulting saturated ammonium sulfate solution and could be collected by centrifugation for 40 min at 10,000 rpm.

Results

Discussion

Conclusion