Instrument to Measure Oxygen Affinity of Hemoglobin

描述（由申请人提供）：正在开发一种测量无细胞血红蛋白氧载体和无细胞血红蛋白/红细胞混合物的氧亲和力的新方法。该方法使用酶从血红蛋白溶液中去除氧，并通过酶动力学计算饱和度。本阶段I SBIR提案的目标是使用该方法开发一种仪器，该仪器将在相关生理条件下快速提供浓缩血红蛋白溶液的氧结合曲线。在特定目标1下，我们将设计和仪器化反应池，以记录使用原儿茶酸（PCA）\原儿茶酸3，4-双加氧酶（PCD）系统对血红蛋白溶液进行酶促脱氧的数据。酶动力学将用于确定氧含量，从而消除对血红蛋白饱和度光谱测量的需要。将对浓缩血红蛋白溶液或与红细胞的混合物进行氧平衡曲线（OEC）测量。在具体目标2下，将开发基于Windows的应用程序，以简化数据采集和电极校准。将使用逐步有限差分方案进行数据分析，该方案根据实验数据计算血红蛋白饱和度。该分析方案将在MATLAB编程环境下实现。数据分析的设计方式将使其在未来能够很容易地纳入Windows应用程序并在线运行。该提议的假设是，目前表征氧亲和力的方法在其无法分析生理相关的浓缩血红蛋白溶液、红细胞悬浮液或红细胞/血红蛋白混合物方面存在固有缺陷，因此需要专门为该任务设计的新仪器。

DESCRIPTION (provided by applicant): A new method to measure the oxygen affinity of cell-free hemoglobin-based oxygen carriers and cell-free hemoglobin/red blood cell mixtures is under development. This method uses an enzyme to remove oxygen from a hemoglobin solution and calculates saturation via enzyme kinetics. The goal of this Phase I SBIR proposal is to use this method to develop an instrument, which will rapidly provide the oxygen binding curves of concentrated hemoglobin solutions under relevant physiologic conditions. Under specific Aim 1, we will design and instrument a reaction cell to record data from the enzymatic deoxygenation of a hemoglobin solution using the protocatechuic acid (PCA)\protocatechuic acid 3,4-dioxygenase (PCD) system. Enzyme kinetics will be used to determine oxygen content, thus eliminating the need for spectral measurements of hemoglobin saturation. Oxygen equilibrium curve (OEC) measurements will be performed on concentrated hemoglobin solutions or in mixtures with red blood cells. Under specific Aim 2, a Windows-based application will be developed to streamline data acquisition and electrode calibrations. Data analysis will be performed using a stepwise finite-difference scheme, which calculates hemoglobin saturation from the experimental data. This analysis scheme will be implemented in the MATLAB programming environment. Data analysis will be designed in such a way that, in the future, it will be easily incorporated into the Windows application and run on-line. The hypothesis underlying this proposal is that current methods to characterize oxygen affinity are inherently flawed in their inability to analyze physiologically-relevant concentrated hemoglobin solutions, red cell suspensions, or red cell/hemoglobin mixtures, thus creating a need for a new instrument designed specially for the task.