DEVELOPMENT OF BIOPHYSICAL METHODS FOR STUDY OF BIO-MOLECULAR REACTIONS

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The utilization of adenosine triphosphate as the prime energy source of many muscle and ion transport reactions is of considerable interest to the understanding of cellular processes. A major problem in studying the pre- steady state kinetics of these reactions has been the lack of ability to follow the disappearance of ATP and the appearance of the reaction products ADP and P04. We have recently utilized near infra-red spectroscopy to detect each of these compounds and have been able to make calibration curves for each in the 1 to 25 micromolar region. Many attributes of this system must be carefully controlled to allow us to have really good reproducibility and sensitivity i.e., approaching that of radiolabeled compounds which utilize P32 labelled ATP. Considerable progress has been made on the problem this year and we now know the measurements can be made. Laser Raman spectroscopy has been carried out on the P04 mixture at pH 11, 9, 6, and 1 as well as pH 7.4 where a mixture of the H2PO4, HP04 occurs but where we get the best spectral signature of ATP, ADP and P04. Much further work will be needed to elucidate the source of the NIR spectra and determine the conditions that affect the spectra. Work on the development of instrumentation for blood substitute continues but a number of technical problems has slowed down the completion of these instruments.