Proteinase assay by capillary electrophoresis employing fluorescence-quenched protein-dye conjugates.
Determination of proteinases--enzymes that catalyze the hydrolysis of peptide bonds--is often difficult due to the presence of interferences in complex biological media and limited sample size. Capillary electrophoresis (CE), with laser-induced fluorescence (LIF) can serve as a useful tool for such determinations. LIF detection offers the advantages of increased sensitivity and increased selectivity. However, direct LIF detection requires the proteinase analyte to be fluorescently derivatized prior to analysis. A viable alternative is offered by the present work, in which protein substrates are first labeled with BODIPY dye, a relatively pH-insensitive, high-fluorescence quantum yield dye. Upon binding of some 4-10 molecules of dye to a single protein, the dye is effectively fluorescence-quenched. Digestion of the BODIPY--labeled and quenched protein by an unlabeled enzyme yields smaller peptide fragments in which the fluorescence of associated BODIPY tags is restored. We will present how the fragmentation pattern of BODIPY-labeled casein changes as a function of incubation time with trypsin, as well as the effect of varying concentrations of trypsin on the BODIPY-casein digest.
Welder
Frank
F
Wake Forest University, Department of Chemistry, P.O. Box 7486, Winston-Salem, NC 27109-7486, USA.
McCorquodale
Elizabeth Moody
EM
Colyer
Christa L
CL
D001896:Boron Compounds; D002364:Caseins / Q000032:analysis / Q000378:metabolism; D019075:Electrophoresis, Capillary / Q000379:methods* / Q000592:standards; D010450:Endopeptidases / Q000378:metabolism*; D005453:Fluorescence; D005456:Fluorescent Dyes; D010449:Peptide Mapping / Q000295:instrumentation / Q000379:methods; D012680:Sensitivity and Specificity; D014357:Trypsin / Q000378:metabolism
C095489:4,4-difluoro-4-bora-3a,4a-diaza-s-indacene; D001896:Boron Compounds; D002364:Caseins; D005456:Fluorescent Dyes; D010450:Endopeptidases; D014357:Trypsin