Thermal inactivation studies of normal and variant human erythrocyte carbonic anhydrases by using a sulphonamide-binding assay

摘要

pHeat-inactivation studies were carried out on the two primary erythrocyte carbonic anhydrase isoenzymes, CA I and CA II, and the secondary isoenzyme of CA I, CA I (+1). In addition, two genetic variants of human isoenzyme CA I, CA Id Michigan (100 Thr→Lys) and CA If London (102 Glu →Lys), and one variant of isoenzyme CA II, CA IIh (251 Asn→Asp), were similarly analysed. The first-order rate constants and Arrhenius plots for these six enzyme forms showed that (1) isoenzyme CA II is more heat-stable than CA I, (2) isoenzyme CA I (+1) is less heat-stable than CA I, (3) the variants CA IIh and CA If London are less heat-stable than the normal enzymes, and (4) isoenzyme CA Id Michigan is more heat-stable than normal CA I. From the values of the slopes of the Arrhenius plots, the energy of activation (iE/isuba/sub) for each isoenzyme and isoenzyme variant was determined, and the following thermodynamic activation parameters were calculated at 55°C: the free energy of activation (ΔiG/isup?/sup), the activation enthalpy (ΔiH/isup?/sup) and the activation entropy (ΔiS/isup?/sup). The ΔiG/isup?/sup for the enzymes shows a relative constancy with compensating variation in ΔiH/isup?/sup and ΔiS/isup?/sup. When the values for ΔiH/isup?/sup are plotted against ΔiS/isup?/sup, an increase in ΔiH/isup?/sup involves a concomitant increase in ΔiS/isup?/sup./p