[Electrophoretic behavior of beta-lactamases in gram-negative bacteria]

The ongoing discoveries of new beta-lactamases, mainly penicillinases, in Gram-negative bacteria has emphasized the problem of their precise identification, and thus their phylogeny. Crude extracts, prepared by sonication, of 14 plasmid beta-lactamases, types TEM, carbenicillinases (CARB or PSE) and oxacillinases (OXA) were analysed by a simple, rapid (3.5 to 4 hours) method of electrophoresis on polyacrylamide (7%) agarose (1.4%) gels, using Tris-glycine buffer at pH 8.7. Preliminary serial dilutions were made to determine enzymic activity levels. Enzymes were then characterized by their relative electrophoretic mobilities. These mobilities had coefficients of variability between 2% and 10%, ranged from 5 to 61, and were correlated with their isoelectric points (pI). Thus, the lower the pI is, the greater the mobility is. Despite the high resolving power of the polyacrylamide-agarose gel system, enzymes with similar pI's and of similar types (PSE-1 and CARB-3, or OXA-1 and OXA-4) or different types (SHV-1 and OXA-6) could not be distinguished on the basis of their mobilities. However, this technique provides for rapid and easy identification of the major penicillinases in Gram-negative bacteria. A combination of polyacrylamide-agarose gel electrophoresis and pH gradient electrophoresis (titration curve) could provide a powerful approach to the study of the molecular structure of these enzymes.