pH Dependence of Solvent Proton Relaxation in Carbonic Anhydrase Solutions: Paramagnetic and Diamagnetic Effects

Abstract

The spin-lattice relaxation rate (T1-1) of solvent water protons has been investigated in solutions of zinc(II) and cobalt(II) bovine carbonic anhydrase B and two alkylated derivatives of the cobalt(II) enzyme. Both modified enzymes contain an acetazolamide residue bridging the metal ion and NT of histjdine-63. At magnetic field strengths corresponding to proton Larmor frequencies of 0.02 and 20 MHz and in the presence of 25 mM Na2SO4, T1-1 is enhanced equally by zinc(II) carbonic anhydrase and the alkylated cobalt(II) enzymes over the pH range from 5 to 12. The enhancement is pH independent below neutrality and drifts upward in a near-linear fashion above pH 8. With cobalt(II) carbonic anhydrase, the enhancement tends toward that of the zinc(II) and alkylated enzymes in the limit of low pH, increases sigmoidally between pH 5 and 8, and drifts upward at higher pH. The data are interpreted to indicate that zinc(II) carbonic anhydrase defines the diamagnetic component of the enhancement caused by the cobalt(II) enzyme and that covalently bound acetazolamide precludes either the presence or the rapid exchange of water from within the coordination sphere of the paramagnetic ion. Subtraction of the diamagnetic contribution from the enhancement obtained with uninhibited cobalt(II) carbonic anhydrase yields a sigmoid-like pH profile. The pattern can be described assuming that a maximal increase in T1-1 requires the basic form of two ionizations exhibiting pkas of 6.11 (standard error=0.03) and 8.5 (standard error=0.4) at the particular buffer concentration used. Approximately 10% of the total paramagnetic enhancement is controlled by the higher pka. Excess acetazolamide or Ethoxzolamide reverses only partially the increase in Y1-1 caused by the substitution of cobalt(II) for zinc(II). The residual paramagnetic enhancement is constant between pH 7.3 and 8.7, and equivalent to approximately 13% of that caused by the free cobalt(II) enzyme in the limit of high pH. © 1979, American Chemical Society. All rights reserved.