DEVELOPMENT OF NIR FOR MONITORING ENZYME-SUBSTRATE REACTIONS

代谢过程对三磷酸腺苷利用的研究一直是 对细胞的整体理解有很大兴趣 活动。近红外光谱最耐人寻味的方面是 在非常快(毫秒)的时间内监控反应的能力 时间刻度。首先，我们必须能够准确地将 三磷酸腺苷水解物和产物。我们已经使用了近红外线 光谱定量ATP、ADP和PI，并成功地 在1至25微摩尔范围内的校准曲线。最近， 这三种磷酸盐的混合物也在 相同的浓度区域。因此，ATP、ADP和PI的区别 可以用近红外光谱分析来实现。我们的下一步是 用近红外和31P跟踪三磷酸腺苷的水解 很巧合。近红外技术成功的必备条件 光谱学是一种精确的校准方法。一旦该校准被 可以对后续的近红外光谱进行比较和量化 与校准过的模型进行比较。在初步研究中，误差百分比 &lt；5%已完成。利用多元线性回归技术， 六个变量的设置被发现产生了与数据最匹配的结果。 这表明可能有几个因素参与了水解酶的作用。 三磷酸腺苷，即三磷酸腺苷-酶复合体和各种其他中间体。

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.