melitten and pyridine-2-azo-4-dimethylaniline-cephalosporin

We developed a dual wavelength spectrophotometric assay that permitted beta-lactamase and beta-galactosidase activities to be measured concurrently in a single sample. We also constructed a target cell, E. coli ML-35p, that was substantially cryptic for its periplasmic beta-lactamase and cytoplasmic beta-galactosidase unless outer membrane (beta-lactamase) or inner membrane (beta-galactosidase) permeabilization occurred. By applying the spectrophotometric assay to whole target cells, we could ascertain the kinetics of inner and outer membrane permeabilization by biological agents, including serum, polymyxin B and mellitin. By monitoring the reactions at an additional wavelength, we could also follow the kinetics of serum-mediated bacteriolysis. These experiments illustrate the principle of multiple wavelength spectrophotometry and provide examples of its use to monitor and dissect the action of biological agents on a gram-negative bacterium.

Topics: Bacteriolysis; beta-Galactosidase; Blood Physiological Phenomena; Cell Membrane Permeability; Cell Wall; Cephalosporins; Chromogenic Compounds; Escherichia coli; Humans; Melitten; Nitrophenylgalactosides; Polymyxin B; Spectrophotometry; Substrate Specificity