Estimation of penicillin concentrations and penicillin metabolising enzyme activities by the Lowry reaction.

Sir: As the present methods for determination of concentrations of penicillins, and activities of penicillin-metabolising enzymes, are not completely satisfactory we examined the possibility of using the LOWRY reaction with penicillins1) for this purpose. All penicillins tested reacted with LOWRY reagent. Table 1 shows that ampicillin, 6-aminopenicillanic acid (6-APA), carbenicillin and several penicilloic acid derivatives reacted strongly. Other penicillins and some derivatives of 4-carboxythiazolidine gave a weaker reaction. The plot of absorbance versus concentration of the reaction with benzylpenicillin and 6-APA is shown in Fig. 1. Under the conditions described by LOWRY et al2), and reading at 500 nm, the curves were sufficiently regular for use as standards. They were sensitive up to 500 ug/ml for 6-APA and to 2,000 ug/ml for benzylpenicillin. Colour developed maximally after about 15 minutes at room temperature and was stable for at least one hour. In enzyme experiments protein was precipitated with 5 % TCA prior to analysis. This slightly depressed colour development. Penicillin acylase was prepared in a manner similar to that already described3) and assayed in a reaction mixture consisting of 550 μmoles of phosphate buffer pH 8, 77 umoles of benzylpenicillin and 2.2 mg of enzyme protein in 11 ml. Incubation time was for 2 hours at 30°C and at the end of this time residual benzylpenicillin was extracted into cold butyl acetate. Samples from the aqueous phase were diluted, brought to a final concentration of 5 % TCA without prior neutralisation, centrifuged and duplicate 1 ml samples used for analysis. The preparation used showed an activity of 23.4 umoles 6-APA formed per mg protein over the 2 hour period. When checked by hydroxylamine analysis4) after neutralisation