title: A Novel Stopped-Flow Assay for Quantitating Carbonic-Anhydrase Activity and Assessing Red-Blood-Cell Hemolysis.
abstract: 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 (kΔpH)-measured via pyranine fluorescence-rises linearly with increases in [bovine CAII] or [murine-RBC lysate]. The y-intercept (no CA) waskΔpH= 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%-causeskΔpHto increase linearly. This y-intercept (0% lysate/100% ostensibly intact RBCs) waskΔpH= 0.0820 s-1, and the maximalkΔpH(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 reduceskΔpHto 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.
authors.affiliation: Department of Physiology and Biophysics, Case Western Reserve University School of Medicine Cleveland, OH, USA.
authors.forename: Pan
authors.initials: P
authors.lastname: Zhao
authors.affiliation: Department of Physiology and Biophysics, Case Western Reserve University School of Medicine Cleveland, OH, USA.
authors.forename: R Ryan
authors.initials: RR
authors.lastname: Geyer
authors.affiliation: Department of Physiology and Biophysics, Case Western Reserve University School of Medicine Cleveland, OH, USA.
authors.forename: Walter F
authors.initials: WF
authors.lastname: Boron
keywords: acid-based balance; bicarbonate; carbon dioxide; out-of-equilibrium solutions; pH-sensitive dyes; phenol red; pyranine