DEVELOPMENT OF NIR FOR MONITORING ENZYME-SUBSTRATE REACTIONS

开发用于监测酶-底物反应的近红外光谱

• 批准号: 3779525
• 负责人: R L BERGER
• 金额: --
• 依托单位: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3779525
• 关键词: adenosine diphosphate; adenosine triphosphate; bioengineering /biomedical engineering; biomedical equipment development; enzyme activity; enzyme substrate complex; infrared spectrometry; phosphorus

The study of ATP utilization by metabolic processes has been of considerable interest to the overall understanding of cellular activities. The most intriguing aspect of near infrared spectroscopy is the capability of monitoring a reaction on a very fast (millisecond) timescale. First of all, we must be able to accurately separate the reactants and products of ATP hydrolysis. We have employed near infrared spectroscopy to quantify ATP, ADP, and Pi and have successfully made calibration curves for each in the 1 to 25 micromolar regime. Recently, mixtures of the three phosphate compounds were also quantified in the same concentration region. Hence, the distinction of ATP, ADP, and Pi can be achieved with near infrared spectroscopy. Our next step has been to follow the hydrolysis of ATP both in the near infrared and with 31P coincidently. A necessary requirement for the success of near infrared spectroscopy is an accurate calibration. Once this calibration is attained, subsequent near infrared spectra can be compared and quantified with the calibrated model. On preliminary studies, percent errors of < 5% have been accomplished. With multiple linear regression techniques, a setting of six variables was found to yield the best fit to the data. This indicates that several factors may be involved in the hydrolysis of ATP, namely ATP-enzyme complexes and various other intermediates.

代谢过程中 ATP 利用率的研究 对细胞的整体理解有很大兴趣 活动。 近红外光谱最有趣的方面是 非常快（毫秒）监控反应的能力 时间尺度。 首先，我们必须能够准确地分离 ATP 水解的反应物和产物。 我们采用了近红外 光谱法定量 ATP、ADP 和 Pi，并成功地 1 至 25 微摩尔范围内每个的校准曲线。 最近， 三种磷酸盐化合物的混合物也进行了定量 相同的浓度区域。 因此，ATP、ADP 和 Pi 的区别 可以通过近红外光谱来实现。 我们的下一步是 在近红外和 31P 下跟踪 ATP 的水解 巧合的是。 近红外成功的必要条件 光谱学是一种精确的校准。 一旦校准完成 获得后，可以比较和量化后续的近红外光谱 与校准模型。 根据初步研究，误差百分比为 < 5% 已完成。 利用多元线性回归技术， 发现六个变量的设置可以产生最适合数据的结果。 这表明，多种因素可能参与了水解过程。 ATP，即ATP-酶复合物和各种其他中间体。