DEVELOPMENT OF NIR FOR MONITORING ENZYME-SUBSTRATE REACTIONS

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Much progress has been made in the quantification of ATP, ADP, and P(1) in a biological reaction, namely Na(+)K(+)ATPase hydrolyzing ATP. Several features of the reactions must be accounted for in order to fully deconvolute the spectra. The water spectrum is the largest hurdle as it is a strong absorber in this region. In addition, the water spectrum is very dependent upon temperature and is affected by the presence of dissolved ions. By obtaining a spectrum of the enzyme within the solution, a effective background is achieved and observation of the hydrolysis of ATP can be accomplished. An error of approximately 10% was found, quite high as compared to the calibration curves of the separate components. Possible contributors to the overall error include dissolved ions, intermediates in the reaction, and protein conformational changes, where it is known that changes in the secondary structure of the protein can alter the vibrational spectrum and thus the near infrared spectrum.