Sensitive detection and study of catalytic species for bimolecular reactions

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Lane, James
Major Professor
Alan W. Schwabacher
Committee Member
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In our lab, we have developed a sensitive and convenient assay for the detection of catalysts for bimolecular reactions. We have shown that catalysis of peptide synthesis by as little as 2.5 fmol (2.5 x 10[superscript]-15 mol) of a damaged enzyme can be detected using our assay. An interesting feature of our procedure is that it requires no complex apparatus, and may be carried out in parallel on many samples. We have used the assay to detect the presence of an enzyme catalyst, however the basic assay design is very versatile and may also be used to identify and evaluate other catalysts for a wide range of useful reactions. At present, sample volumes of 5 ul are studied and may be possible to lower this reaction volume substantially. The nature of the assay allows for the simultaneous screening of catalysts under varying reaction conditions;The original idea was to develop the assay and use it to screen for new catalytic antibodies with peptide ligase activity. Unfortunately however, no catalytic antibodies were obtained even using traditional screening and characterization methods. Nevertheless, the potential of the assay for screening large antibody pools has been demonstrated by the detection of solvent damaged [alpha]-chymotrypsin catalyzed peptide bond formation. The activity of this catalyst is comparable to that expected for first generation catalytic antibodies;One factor contributing to the high sensitivity of the assay is the extremely large association constant between avidin and biotin. This property has also been taken advantage of by attaching biotin to 7-azatryptophan the latter of which serves as a fluorescent probe owing to its unique excitation and emission spectra. The biotin tag causes 7-azatryptophan to be held in close proximity to the protein avidin and by monitoring changes in the emission spectrum of 7-azatryptophan, information has been obtained about the motional constraints felt by small molecules near the surfaces of proteins.

Sat Jan 01 00:00:00 UTC 1994