The design, synthesis and evaluation of active site directed, fluorescent, peptidylchloromethane affinity labels of proteinases

dc.contributor.committeeMemberDyckes, Douglas F.
dc.contributor.committeeMemberCox, James R., Jr.
dc.contributor.committeeMemberKohn, Harold Lewis
dc.contributor.committeeMemberDeming, Stanley N.
dc.contributor.committeeMemberDudkiewicz, Alan B.
dc.creatorPenny, Glenn Stanley
dc.description.abstractA series of N- -l-dimethylamino-5-napthalenesulfonyl (dansyl) derivatives of Peptidylchloromethanes (chloromethyl ketones) were synthesized and employed to introduce the fluorescent dansyl moiety specifically into the active sites of proteinases via affinity labelling. Dansylalanyllysylchloromethane (DALCM) was utilized to inactivate and fluorescently label trypsin and the trypsin-like enzymes, thrombin and acrosin. Dansylleucylphenylalanylchloromethane (DLPCM) was synthesized and selectively employed as an inhibitor of chymotrypsin. The di, tri, and tetrapeptides - dansylprolylalanylchloromethane (DPACM) dansylalanylprolylalanylchloromethane (DAPACM), and dansylprolylalanylprolylalanylchloromethane (DPAPACM) - were synthesized and their interaction with elastase evaluated. These fluorescent, fast acting proteinase inhibitors possess enormous potential for the localization, isolation and characterization of enzymes. DALCM, for example, has been used to localize acrosin on the head of spermatozoa. An additional benefit of DALCM lies in its markedly fast rate of trypsin inactivation. This permits the practical utilization of stoichiometric amounts of inhibitor to achieve selective 100% trypsin inactivation even at high dilutions. The transfer of energy between the tryptophan residues of proteinases and a specifically placed dansyl group was used to evaluate proximity relationships in dansylated inhibitor-enzyme ,conjugates of trypsin, chymotrypsin, elastase and thrombin. The anomalies between the mean tryptophan-dansyl distances calculated from fluorescence measurements and the expected mean distances based on x-ray data were explored. The energy transfer studies herein reported provide positive evidence for the conformational homology of a-chymotrypsin and thrombin. A fluorometric enzyme inactivation assay based on energy transfer was developed and employed to determine the rate of inactivation of proteinases by dansylated peptidyl chloromethanes. In this assay the tryptophan residues of the proteinase are excited at 295 nm and the dansyl emission of the developing fluorescent enzyme-inhibitor conjugate is monitored as a function of time.
dc.description.departmentChemistry, Department of
dc.format.digitalOriginreformatted digital
dc.rightsThis item is protected by copyright but is made available here under a claim of fair use (17 U.S.C. Section 107) for non-profit research and educational purposes. Users of this work assume the responsibility for determining copyright status prior to reusing, publishing, or reproducing this item for purposes other than what is allowed by fair use or other copyright exemptions. Any reuse of this item in excess of fair use or other copyright exemptions requires express permission of the copyright holder.
dc.subjectChloromethyl ketone
dc.subjectEnergy transfer
dc.subjectFluorometric enzyme inactivation assay
dc.subjectEnzyme localization
dc.subjectFluorescent affinity label
dc.subjectTrypsin inhibitor
dc.titleThe design, synthesis and evaluation of active site directed, fluorescent, peptidylchloromethane affinity labels of proteinases
dc.type.genreThesis of Natural Sciences and Mathematics, Department of of Houston of Philosophy


Original bundle

Now showing 1 - 1 of 1
Thumbnail Image
4.61 MB
Adobe Portable Document Format