PHOTOAFFINITY LABELS BY LASER SPECTROSCOPY

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The major objective of this work is to provide the knowledge necessary for sound application and interpretation of photolabeling experiments. A major objective in the study of living systems is to associate function with chemical structure. Several experimental approaches have been developed to facilitate this goal. Among the most useful of these are photolabeling procedures. In this approach an activatable reagent is positioned adjacent to a target structure. This is often accomplished by taking advantage of the high affinity of the target for a particular substrate. The key steps in the labeling process involve activation of the latent reactivity of the reagent by irradiation with light and covalent bond formation between the reagent and the target biological structure. Once this has been accomplished, the target structure can be broken into smaller parts and the linked label located. The aim of the research in this application is to examine the details of activation of the photolabeling agent by light, study the process of covalent bond formation, develop new photochemicals useful as labels, and test these findings in actual biological systems. A number of methods will be employed to accomplish these goals. However, the centerpiece of our approach is the application of time-resolved laser transient absorption spectroscopy to this problem. This method allows the direct observation of the photolabeling process and provides detailed information on the key activation and bonding steps. The laser spectroscopic results will be combined with the results of other, more classical, experiments to accomplish the aims of this reasearch.