DEVELOPMENT OF NIR FOR MONITORING ENZYME-SUBSTRATE REACTIONS

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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.