Capillary electrophoresis with laser-induced fluorescence detection as a tool for enzyme characterization and inhibitor screening.
An effective, rapid and economical CE/LIF (capillary electrophoresis/laser-induced fluorescence) method was developed and applied to the characterization of signal peptidase (SPase) enzyme, which is a target for the screening of new drug candidates. In this method, CE separates the product from the substrate and LIF selectively detects the fluorescence-labeled product and substrate. By measuring the increase of the product as a function of time, one can monitor the progression of the enzyme reaction. The progression curves were also used for screening inhibitors for this enzyme. The effects of various reaction conditions were also studied and discussed. In addition, this CE/LIF method was applied to the determination of the enzyme activity, the quality control of the substrate and/or enzymes, and the cross-reactivity of inhibitors to the enzyme. It can be concluded that this method is suitable for high throughput screening (HTS) assays because it can deliver fast, sensitive, quantitative, and reliable results.
Liu
Dan-Ning
DN
School of Pharmaceutical Science and Technology, Tianjin University, Tianjin, China.
Li
Li
L
Lu
Wei-Ping
WP
Liu
Yao-Qing D
YQ
Wehmeyer
Ken R
KR
Bao
James J
JJ
D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D004353:Drug Evaluation, Preclinical; D019075:Electrophoresis, Capillary / Q000295:instrumentation* / Q000379:methods; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000737:chemistry* / Q000494:pharmacology; D005453:Fluorescence; D006863:Hydrogen-Ion Concentration; D007834:Lasers; D008027:Light; D008565:Membrane Proteins / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000528:radiation effects; D010446:Peptide Fragments / Q000737:chemistry; D012680:Sensitivity and Specificity; D012697:Serine Endopeptidases / Q000032:analysis* / Q000528:radiation effects; D013050:Spectrometry, Fluorescence / Q000295:instrumentation / Q000379:methods; D013329:Structure-Activity Relationship; D013997:Time Factors; D014508:Urea / Q000494:pharmacology
D004791:Enzyme Inhibitors; D008565:Membrane Proteins; D010446:Peptide Fragments; D014508:Urea; D012697:Serine Endopeptidases; C026515:type I signal peptidase; D004121:Dimethyl Sulfoxide