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.

代谢过程对ATP利用的研究已有 对细胞的整体理解非常感兴趣 活动。 近红外光谱的最吸引人的方面是 在非常快（毫秒）上监测反应的能力（毫秒） 时间尺度。 首先，我们必须能够准确地分开 ATP水解的反应物和产物。 我们已经雇用了红外线 量化ATP，ADP和PI的光谱法，并已成功制作 在1至25微摩尔制度中的校准曲线。 最近， 在 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法实现。 我们的下一步是 遵循近红外和31p的ATP的水解 巧合。 近红外成功的必要要求 光谱是准确的校准。 一旦这种校准 达到的，可以比较并定量随后的红外光谱 使用校准模型。 关于初步研究，百分比的错误 <5％已完成。 使用多个线性回归技术， 发现六个变量的设置可为数据带来最佳拟合。 这表明可能有几个因素参与 ATP，即ATP-酶复合物和其他各种中间体。