A colorimetric procedure for measuring beta-lactamase activity

The enzymatic hydrolysis of benzylpenicillin was measured by a novel colorimetric procedure. The penicilloic acid generated from the hydrolysis of penicillin was reacted with CuSO4 and neocuproine to form a colored complex having a maximal absorption at 454.5 nm. A plot of absorbance versus beta-lactamase activity yielded a straight line from 1 to 5 mU of enzyme. Using TEM-1 as the model beta-lactamase, a Km of 46 microM was observed with benzylpenicillin serving as the substrate. When the assay was used to determine levels of benzylpenicillin, the absorbance was found to be linearly proportional to exogenously added penicillin from 2.8 to 88 microM. This procedure is simple to use and can be employed to measure the hydrolysis of other beta-lactam antibiotics.