A microtiter colorimetric assay for the HIV-1 protease.
We have developed a novel colorimetric assay for the HIV-1 protease that is suitable for high-throughput screening of inhibitors. This assay utilizes two nonenzymatic reaction steps, which are carried out in succession following enzymatic hydrolysis of a synthetic peptide. The first step involves a carbamylation reaction between cyanate and the nascent alpha amino group resulting from enzymatic hydrolysis. The second step involves a carbamidodiacetyl reaction between 2,3-butanedione monoxime (diacetylmonoxime) and the de novo carbamido compound. The entire assay can be performed in a microtiter plate and is amenable to automation. In addition, this peptidolysis assay is readily adaptable to other proteolytic enzymes and their substrates.
Stebbins
J
J
Department of Molecular Genetics, SmithKline Beecham Pharmaceuticals, King of Prussia, Pennsylvania 19406, USA.
Debouck
C
C
D000595:Amino Acid Sequence; D002863:Chromogenic Compounds; D003124:Colorimetry / Q000379:methods*; D003931:Diacetyl / Q000031:analogs & derivatives; D004353:Drug Evaluation, Preclinical; D016333:HIV Protease / Q000032:analysis* / Q000378:metabolism; D017320:HIV Protease Inhibitors / Q000737:chemistry / Q000494:pharmacology; D007202:Indicators and Reagents; D007700:Kinetics; D015394:Molecular Structure; D009842:Oligopeptides / Q000737:chemistry; D013379:Substrate Specificity
D002863:Chromogenic Compounds; D017320:HIV Protease Inhibitors; D007202:Indicators and Reagents; D009842:Oligopeptides; C004717:diacetylmonoxime; D016333:HIV Protease; D003931:Diacetyl