Zhao Pan, Geyer R Ryan, Boron Walter F
Department of Physiology and Biophysics, Case Western Reserve University School of Medicine Cleveland, OH, USA.
Front Physiol. 2017 Mar 28;8:169. doi: 10.3389/fphys.2017.00169. eCollection 2017.
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/44 mM [Formula: see text]/pH 8.41, to generate an out-of-equilibrium CO/[Formula: see text] solution containing ~0.5% CO/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 → CO + HO. Proof-of-concept studies (no intact RBCs) show that the pH-relaxation rate constant ()-measured via pyranine fluorescence-rises linearly with increases in [bovine CAII] or [murine-RBC lysate]. The y-intercept (no CA) was = 0.0183 s. 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 to increase linearly. This y-intercept (0% lysate/100% ostensibly intact RBCs) was = 0.0820 s, and the maximal (100% lysate/0% intact RBCs) was 1.304 s. 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 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.
我们报告了一种新型碳酸酐酶（CA）测定法及其在停流（SF）实验中用于定量红细胞（RBC）裂解的应用。我们将两种盐溶液混合，一种含有HEPES/pH 7.03，另一种含有约1% CO/44 mM [化学式：见正文]/pH 8.41，以在SF反应池中生成一种含有约0.5% CO/22 [化学式：见正文]/pH约7.25（10°C）的非平衡CO/[化学式：见正文]溶液。CA催化细胞外pH松弛至约7.50：[化学式：见正文] + H → CO + HO。概念验证研究（无完整RBC）表明，通过吡喃荧光测量的pH松弛速率常数（）随[牛CAII]或[鼠RBC裂解物]的增加呈线性上升。y轴截距（无CA）为 = 0.0183 s。将越来越多的鼠RBC裂解物与表面上完整的RBC（SF前溶血≅0.4%）混合，在反应池中将总[血红蛋白]固定在2.5 μM以模拟从表面上0%到100%的溶血，导致增加呈线性。该y轴截距（0%裂解物/100%表面上完整的RBC）为 = 0.0820 s，最大（100%裂解物/0%完整RBC）为1.304 s。因此，反应池中平均溶血百分比约为4.9%。酚红吸光度测定产生难以区分的结果。从0.4%增加到4.9%可能反映了快速混合过程中RBC的机械破坏。在所有荧光研究中，CA阻滞剂乙酰唑胺将降低至接近未催化的值，这意味着所有CA活性都是细胞外的。我们的裂解测定法简单、灵敏且精确，对于校正生理SF实验中的裂解效应将很有价值。应用于血浆、组织培养基和器官灌注液的基础CA测定法可在各种应用中评估裂解情况。
