A spectrophotometric method for the determination of creatine phosphokinase and myokinase.

where ATP = adenosine triphosphate and ADP= adenosine diphosphate. The substances most commonly determined have been creatine or creatine phosphate; e.g. Banga (1943), Askonas (1951), Narayanaswami (1952), Ennor & Rosenberg (1954), Kuby, Noda & Lardy (1954) and Chappell & Perry (1954). These methods may not always be convenient for the determination of creatine phosphokinase activity in tissue homogenates, since the amount of tissue used in reaction mixtures makes the introduction of significant amounts of endogenous substances a distinct possibility. Themethod described here, although not more sensitive than the methods based on the colorimetric estimation of creatine, makes it possible to measure enzymic activities in tissue homogenates and extracts diluted 2000 to 20 000 times, and to follow continuously the time course of the reaction in a total volume of about 4 ml. Under these conditions effects due to endogenous substances are negligible. The method is based on Kornberg's assay procedure for ATP (Kornberg, 1950), whereby the formation of ATP from ADP and creatine phosphate is linked to the reduction of triphosphopyridine nucleotide (TPN). When the rate-limiting step in the reaction sequence is that catalysed by creatine phosphokinase, spectrophotometric measurement of the rate of reduction ofTPN gives the rate ofATP formation, and thus the activity of the enzyme. The method has also been applied to the determination of myokinase activity by measuring the rate of formation ofATP from ADP. The methods for the assay of myokinase due to Kalckar (1943, 1947) are laborious. The most sensitive method is probably that based on the firefly luminescence system for the estimation of ATP (Strehler & Totter, 1952), but is not convenient for general use. The coupled activities of myokinase and creatine phosphokinase have been used by Chappell & Perry (1954) to assay myokinase. The present method has the advantage of convenience over most of the older methods. The reactions are followed continuously in the spectrophotometer, and the rates catalysed by small amounts of tissue can be measured with ease. An outline of the method has been given elsewhere (Oliver, 1954). Complete experimental details are included here.