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.