LOW TEMPERATURE CHEMISTRY/KINETICS/PHOTOAFFINITY LABELS

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We intend to covalently modify a-chymotrypsin with various aryl azides and diazocompounds. Crystals of the pure modified proteins will be photolyzed at -196 degrees. We will use ESR spectroscopy to directly detect the resultant enzyme bound nitrenes and carbenes. We will use ESR to measure the kinetics of reaction of the nitrenes and carbenes with the enzyme. We expect the reaction will involve hydrogen atom transfer by quantum mechanical tunneling to give a radical-pair. The kinetic barrier will be fit to an Eckart barrier to obtain the barrier width-the distance travelled by the tunneling hydrogen from an amino acid residue to the carbene or nitrene. We will detect the product radical pair by ESR. The spectrum of the radical pair will give a second measure of the separation of the pair. The nuclear hyperfine interaction present in the pair will allow us to identify the amino acid residue which is the hydrogen donor. We will study the products of the low temperature photochemistry. It is proposed that cryogenic conditions will improve the labelling efficiency. If successful this project will produce an important new method in photoaffinity labelling for identifying constituents of the active sites of biomolecues.