A simple and sensitive method for the assay of trans-cinnamic acid 4-hydroxylase by use of tritiated substrate is described. This method is based on the migration of tritium during the enzyme-catalyzed hydroxylation. The hydroxylase activity is detected in microsomes from Phaseohs mungo.
The tritium method can be used practically with sensitivity similar to that of the 14C method. In view of the time and labor required the tritium method is obviously more advantageous.
[runs-Cinnamic acid 4-hydroxylase (no assignment) catalyzes the conversion of trans-cinnamic acid to 4-hydroxycinnamic acid. This reaction is the initial hydroxylating step in the biosynthetic pathway toward lignin and flavonoids (1,2) from trans-cinnamic acid. The enzyme activity has been detected in various higher plants (3-12) and a fungus (13). In these studies the activity was assayed by the 14C method, which included extraction, concentration, and paper chromatographic isolation of the product. This method is sensitive but rather laborious and time consuming. To overcome these difficulties it was hoped that a new assay method fortrans-cinnamic acid 4-hydroxylase activity could be developed. In the present study, I modified the tritium method developed for the assay of phenylalanine 4-hydroxylase (EC 1.14.16.1) ( 14), since Russell et al. ( 15) had reported that the tritium label of truns-[4-3H]cinnamic acid shifted to the 3 or 5 position during 4-hydroxylation of the substrate. After hydroxylation, tritium of the product, 4-hydroxycinnamic acid, was released by iodination, and the resulting tritium water was measured for the trans-cinnamic acid 4-hydroxylase activity. This assay system was examined by use of microsomes of the mung bean, Phaseolus mungo.
MATERIALS AND METHODS
Materials. L-[4-"H]Phenylalanine
(12 Ci/mmol) was purchased from Radiochemical Centre (Amersham, England), and truns-[3-14C]cinnamic acid (50 mCi/mmol) was purchased from Schwarz/Mann Co. (New York). NADPH was obtained from Oriental Yeast Co.