AUTHOR=Zhao Pan , Geyer R. Ryan , Boron Walter F. 

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

JOURNAL=Frontiers in Physiology

VOLUME=8

YEAR=2017

URL=https://www.frontiersin.org/journals/physiology/articles/10.3389/fphys.2017.00169

DOI=10.3389/fphys.2017.00169

ISSN=1664-042X

ABSTRACT=<p>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% CO<sub>2</sub>/44 mM <inline-formula><mml:math id="M1" xmlns:mml="http://www.w3.org/1998/Math/MathML"><mml:msubsup><mml:mrow><mml:mtext>HCO</mml:mtext></mml:mrow><mml:mrow><mml:mn>3</mml:mn></mml:mrow><mml:mrow><mml:mo>-</mml:mo></mml:mrow></mml:msubsup></mml:math></inline-formula>/pH 8.41, to generate an out-of-equilibrium CO<sub>2</sub>/<inline-formula><mml:math id="M2" xmlns:mml="http://www.w3.org/1998/Math/MathML"><mml:msubsup><mml:mrow><mml:mtext>HCO</mml:mtext></mml:mrow><mml:mrow><mml:mn>3</mml:mn></mml:mrow><mml:mrow><mml:mo>-</mml:mo></mml:mrow></mml:msubsup></mml:math></inline-formula> solution containing ~0.5% CO<sub>2</sub>/22 <inline-formula><mml:math id="M3" xmlns:mml="http://www.w3.org/1998/Math/MathML"><mml:msubsup><mml:mrow><mml:mtext>HCO</mml:mtext></mml:mrow><mml:mrow><mml:mn>3</mml:mn></mml:mrow><mml:mrow><mml:mo>-</mml:mo></mml:mrow></mml:msubsup></mml:math></inline-formula>/pH ~7.25 (10°C) in the SF reaction cell. CA catalyzes relaxation of extracellular pH to ~7.50: <inline-formula><mml:math id="M4" xmlns:mml="http://www.w3.org/1998/Math/MathML"><mml:msubsup><mml:mrow><mml:mtext>HCO</mml:mtext></mml:mrow><mml:mrow><mml:mn>3</mml:mn></mml:mrow><mml:mrow><mml:mo>-</mml:mo></mml:mrow></mml:msubsup></mml:math></inline-formula> + H<sup>+</sup> → CO<sub>2</sub> + H<sub>2</sub>O. Proof-of-concept studies (no intact RBCs) show that the pH-relaxation rate constant (<italic>k</italic><sub>ΔpH</sub>)—measured via pyranine fluorescence—rises linearly with increases in [bovine CAII] or [murine-RBC lysate]. The y-intercept (no CA) was <italic>k</italic><sub>ΔpH</sub> = 0.0183 s<sup>−1</sup>. 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 <italic>k</italic><sub>ΔpH</sub> to increase linearly. This y-intercept (0% lysate/100% ostensibly intact RBCs) was <italic>k</italic><sub>ΔpH</sub> = 0.0820 s<sup>−1</sup>, and the maximal <italic>k</italic><sub>ΔpH</sub> (100% lysate/0% intact RBCs) was 1.304 s<sup>−1</sup>. 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 <italic>k</italic><sub>ΔpH</sub> 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.</p>