Chromogenic redox assay for beta-lactamases yielding water-insoluble products. I. Kinetic behavior and redox chemistry.

We describe a chromogenic detection system for beta-lactamase which yields water-insoluble colored products. The assay is based on kinetic measurement of the appearance of color due to the beta-lactamase-initiated redox reaction. The substrates are C3' thiolate-substituted cephalosporins, which, after enzyme-catalyzed hydrolysis of the beta-lactam ring, undergo elimination of the thiolate ion. This thiolate, in a postenzymatic step, reduces the tetrazolium salts, which are water-soluble colorless compounds, to a colored water-insoluble precipitate of formazan. Our model in this study was a beta-lactamase Enterobacter cloacae P-99-catalyzed reaction of thiolacetate cephalosporin with several tetrazolium salts. We found that the reaction rate is dependent on the concentration of the electron carrier 5-methyl phenazinium methyl sulfate, the pKa of the C3' thiolate substituent of the cephalosporin substrate, and the reduction potential of the tetrazolium salts. A kinetic study of this system yielded a rate law for the reaction. We present a mechanism of the reaction and determination of the kinetic parameters for the process. The sensitivity of this kinetic assay is very high; we detect 3 x 10(-10) M beta-lactamase P-99, which is approximately 30 mIU. The assay times are very short, lasting from 2 to 5 min. The new assay system is particularly suitable for a rapid detection of beta-lactamases in bacterial colonies and in enzyme immunoassays where beta-lactamase may be used as the label.