The rapid fixed-time assay for penicillinase was modified for measuring β-lactamase activity with twelve substrates, i.e., benzylpenicillin, ampicillin, cloxacillin, methicillin, carbenicillin, cefazolin, cephalothin, cephaloglycin, cephalexin, cephalosporin C, 7-aminocephalosporanic acid, and cefoxitin. The method depends upon the reduction of iodine by the hydrolyzed substrate. Determined experimentally, 1 mol of hydrolyzed penicillins consumed 3.4 to 4.0 mol of iodine (I
2
). Iodine consumption of hydrolyzed cephalosporins varied widely from 1.7 for cephalothin to 3.7 for cefazolin. The method is useful for routine assay of β-lactamase activity with various substrates.
The penicillinase from an Escherichia coli strain harboring an R factor RGN823 was purified and its properties were compared with those of a known type I penicillinase mediated by R factors. The molecular weight and S20,W of the enzyme were 22,600 and 2.42S, respectively. The isoelectric point of the enzyme was 6.9. These values are clearly different from those of type I penicillinase. The specific activity of the enzyme was 84,700 units per mg of the purified enzyme protein, which is about 20 times higher than that of the type I penicillinase. However, similarities were observed between the enzyme and the type I-penicillinase at optimal pH (6.5 to 7.0), optimal temperature (40 to 45C), substrate specificity, Michaelis constants for penicillins and cephaloridine, and effect of inhibitors. Furthermore, antiserum against type I penicillinase showed cross-reaction against this enzyme. The enzyme was named type lb penicillinase, and the original type I penicillinase was renamed type la-penicillinase.
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