Gryczynski Z, Gering H, Bucci E
Department of Biochemistry and Molecular Biology, University of Maryland Medical School, Baltimore 21201, USA.
Anal Biochem. 1998 Jan 15;255(2):176-82. doi: 10.1006/abio.1997.2483.
A cell was constructed in order to study hemoglobin's reaction with gaseous ligands. The temperature of the hemoglobin sample is systematically altered within a given temperature range (275-310 degrees K), while the percentage of oxygen in the equilibrating gas is kept constant. The equilibration time of the sample at each temperature step depends on sample concentration, ligand affinity, and absolute temperature; in most cases, the equilibration time is on the order of minutes. The construction of the optical compartment allows the experimenter to vary the optical pathlength using specially designed spacers, thus making it possible to study hemoglobin-ligand interactions over a wide range of protein concentrations (0.1-200 mg/ml). Optical glass is used in the construction of the cuvette in order to optimize its optical stability over a long period of time. At equilibrium the absorption spectrum of the sample is collected and decomposed into the relative contributions of oxy-Hb, deoxy-Hb, and ferric-Hb, thus revealing the fraction of oxyhemoglobin as well as any baseline drifts and protein degradation. Temperature steps of 1 degree K are already sufficient to change the absorption spectra in a significant way. This type of setup is also advantageous in that the experimenter can change the sample at any point (temperature) without having to restart the entire experiment. This makes it possible to study the oxygen binding characteristics of unstable hemoglobins. Analyses of the binding curves obtained with this technique immediately yield the overall oxygen binding constants beta i together with the respective standard enthalpies delta H(i).
构建了一个细胞用于研究血红蛋白与气态配体的反应。在给定温度范围（275 - 310开尔文）内系统地改变血红蛋白样品的温度，同时使平衡气体中的氧气百分比保持恒定。在每个温度步骤下样品的平衡时间取决于样品浓度、配体亲和力和绝对温度；在大多数情况下，平衡时间为几分钟量级。光学隔室的构造使实验者能够使用特殊设计的间隔物改变光程长度，从而能够在很宽的蛋白质浓度范围（0.1 - 200毫克/毫升）内研究血红蛋白 - 配体相互作用。比色皿的构造中使用光学玻璃以在长时间内优化其光学稳定性。在平衡时，收集样品的吸收光谱并分解为氧合血红蛋白、脱氧血红蛋白和高铁血红蛋白的相对贡献，从而揭示氧合血红蛋白的比例以及任何基线漂移和蛋白质降解情况。1开尔文的温度步长就足以显著改变吸收光谱。这种类型的设置还有一个优点，即实验者可以在任何点（温度）更换样品而无需重新开始整个实验。这使得研究不稳定血红蛋白的氧结合特性成为可能。用该技术获得的结合曲线分析可立即得出总的氧结合常数βi以及各自的标准焓ΔH(i)。
