A spectrophotometric assay for trans-cinnamic acid 4-hydroxylase activity

trans-Cinnamic acid 4-hydroxylase (trans-cinnamic acid, NADPH: oxygen oxidoreductase [4-hydroxylating]) can be rapidly and precisely assayed by the spectrophotometric measurement of the production of 4-hydroxy-trans-cinnamic acid at 340 nm after acidification of the reaction mixture and subsequent readjustment to pH 11. For the assay of crude extracts and other preparations with high intrinsic absorption at 340 nm, the assay can be modified by extraction of the 4-hydroxy-trans-cinnamic acid from the acidified assay mixture through diethyl ether into alkali before spectrophotometric estimation. trans-Cinnamic acid 2-hydroxylase can be routinely detected and assayed in the same extract.