A MICROTITER COLORIMETRIC ASSAY FOR THE HIV-1 PROTEASE

We have developed a novel colorimetric assay for the HIV-1 protease th at is suitable for high-throughput screening of inhibitors. This assay utilizes two nonenzymatic reaction steps, which are carried out in su ccession following enzymatic hydrolysis of a synthetic peptide. The fi rst step involves a carbamylation reaction between cyanate and the nas cent alpha amino group resulting from enzymatic hydrolysis. The second step involves a carbamidodiacetyl reaction between 2,3-butanedione mo noxime (diacetylmonoxime) and the de novo carbamido compound. The enti re assay can be performed in a microtiter plate and is amenable to aut omation. In addition, this peptidolysis assay is readily adaptable to other proteolytic enzymes and their substrates. (C) 1997 Academic Pres s.