81Br-nmr studies of carbonic anhydrase

Abstract

81Br nmr measurements have been made on high (bovine; BCA) and low (human-B; HCAB) specific activity forms of carbonic anhydrase and on a chemically modified form of the human enzyme (carboxyamidomethyl; CAM-HCAB). The high specific activity form of the enzyme, BCA, exhibits a 81Br line broadening which is determined by the lifetime of Br − bound to the zinc ion of the enzyme. The low specific activity form of the enzyme, HCAB, under similar conditions of concentration, pH, etc., does not exhibit a 81Br nmr line broadening. Cl − Br − competitive binding studies, using 35Cl nmr, suggests that the failure to observe 81Br broadening is due to an increase in the lifetime of a zinc bound Br −. An increase in this lifetime by a factor of 10–100 over that exhibited by BCA is sufficient to abolish the line broadening. A modified form of HCAB, CAM-HCAB, does, however, exhibit a 81Br nmr line broadening. Estimates of the lifetime of zinc bound Br −, τ M, are 4 × 10 −7 sec. for CAM-HCAB at pH 8 and 1 × 10 −7 sec. for BCA at pH 7. The lifetime for Br − bound to HCAB is estimated to be ≥10 −6 sec.