Micro-iodometric assay for penicillinase

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Abstract
A sensitive method for measuring the rate of hydrolysis of penicillin to penicilloic acid by penicillinase is described. It depends upon the reduction of I by penicilloic acid but not by penicillin, and it is carried out by measuring the rate of decolorization of the blue starch-iodide complex when enzyme and substrate react in the presence of starch-iodide. The method was developed for the study of penicillinase reaction kinetics; reaction rates were measured for penicillin concentrations between 1 and 800 [mu][image] and for enzyme activities between 0-001 and 5 [mu][image]/ml/hr. The micro-assay is 1000 times as sensitive as existing chemical methods, and despite non-specific iodine absorption was useful for the detection and measurement of small amounts of penicillinase in complex mixtures such as broth. Staphylococcal penicillinase was apparently not affected by starch or by I under the conditions of the assay, and the method was used for the measurement of the Michaelis constants (Km) of the enzyme with various substrates.