A Novel Stopped-Flow Assay for Quantitating Carbonic-Anhydrase Activity and Assessing Red-Blood-Cell Hemolysis

We report a novel carbonic-anhydrase (CA) assay and its use for quantitating red-blood-cell (RBC) lysis during stopped-flow (SF) experiments. We combine two saline solutions, one containing HEPES/pH 7.03 and the other, ~1% CO₂/44 mM [Formula: see text]/pH 8.41, to generate an out-of-equilibrium CO₂/[Formula: see text] solution containing ~0.5% CO₂/22 [Formula: see text]/pH ~7.25 (10°C) in the SF reaction cell. CA catalyzes relaxation of extracellular pH to ~7.50: [Formula: see text] + H⁺ → CO₂ + H₂O. Proof-of-concept studies (no intact RBCs) show that the pH-relaxation rate constant (k_ΔpH)-measured via pyranine fluorescence-rises linearly with increases in [bovine CAII] or [murine-RBC lysate]. The y-intercept (no CA) was k_ΔpH = 0.0183 s^-1. Combining increasing amounts of murine-RBC lysate with ostensibly intact RBCs (pre-SF hemolysis ≅0.4%)-fixing total [hemoglobin] at 2.5 μM in the reaction cell to simulate hemolysis from ostensibly 0 to 100%-causes k_ΔpH to increase linearly. This y-intercept (0% lysate/100% ostensibly intact RBCs) was k_ΔpH = 0.0820 s^-1, and the maximal k_ΔpH (100% lysate/0% intact RBCs) was 1.304 s^-1. Thus, mean percent hemolysis in the reaction cell was ~4.9%. Phenol-red absorbance assays yield indistinguishable results. The increase from 0.4 to 4.9% presumably reflects mechanical RBC disruption during rapid mixing. In all fluorescence studies, the CA blocker acetazolamide reduces k_ΔpH to near-uncatalyzed values, implying that all CA activity is extracellular. Our lysis assay is simple, sensitive, and precise, and will be valuable for correcting for effects of lysis in physiological SF experiments. The underlying CA assay, applied to blood plasma, tissue-culture media, and organ perfusates could assess lysis in a variety of applications.