Instrument to Measure Oxygen Affinity of Hemoglobin

描述(申请人提供)：正在开发一种新的方法来测量基于无细胞血红蛋白的氧气载体和无细胞血红蛋白/红细胞混合物的氧亲和力。这种方法使用一种酶来去除血红蛋白溶液中的氧气，并通过酶动力学计算饱和度。此一期SBIR方案的目标是利用该方法开发一种仪器，该仪器将快速提供相关生理条件下浓缩血红蛋白溶液的氧结合曲线。在具体目标1下，我们将设计和测量一个反应室，以记录原儿茶酸(PCA)-原儿茶酸3，4-双加氧酶(PCD)系统在血红蛋白溶液中的酶促脱氧数据。将使用酶动力学来确定氧含量，从而消除了对血红蛋白饱和度进行光谱测量的需要。氧平衡曲线(OEC)测量将在浓缩的血红蛋白溶液或与红细胞的混合物中进行。在具体目标2下，将开发一个基于Windows的应用程序，以简化数据采集和电极校准。数据分析将使用逐步有限差分方案进行，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。数据分析将以这样一种方式设计，在未来，它将很容易地合并到Windows应用程序中并在网上运行。这一提议背后的假设是，目前表征氧亲和力的方法存在固有的缺陷，无法分析与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，因此需要专门为这项任务设计的新仪器。

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.