Liver phenylalanine hydroxylase assay.

The first reaction is catalyzed by phenylalanine hydroxylase and the second reaction, which generates the reduced form of pteridine cofactor (biopterin), is catalyzed by dihydropteridine reductase (l-3). A direct assay of phenylalanine hydroxylase can be achieved by supplying optimal concentrations of reduced pteridine cofactor or an analog of the reduced cofactor (6,7-dimethyl-5,6,7,8-tetrahydropterine) maintained in its reduced state by the addition of NADH or NADPH (4). The assay avoids the need for an accessory NADPH enzyme-generating system and ensures that the only rate-determining component in the hydroxylating reaction is the concentration of phenylalanine hydroxylase. A typical assay for phenylalanine hydroxylase is given in Table 1. The control flasks contain all of the reagents except L-phenylalanine. Incubations are carried out at 37” in a 1Oml Erlenmeyer flask in a Dubnoff shaker, in air, and the reaction is started by the addition of enzyme to the control and experimental flasks. The shaking rate is 140 oscillations per minute. The rate of the reaction was linear with time for at least 30 min and is proportional to enzyme concentration. After 8 min of incubation the reaction mixtures are deproteinized with 0.1 ml of 20% trichloroacetic acid, to stop the reaction, and placed in ice for 5 min. The acidified contents of the flasks are centrifuged at 3000g for 5 min and 0.5 ml of the