Date of this Version
Various investigations related to the biosynthesis of o-hydroxycinnamic acid in sweetclover require that simple, rapid methods of assay be available for both the cis isomer (coumarinic acid) and the trans isomer (o-coumaric acid). This report deals with two such methods, both of which represent refinements of a technique for the fluorometric assay of coumarin described in an earlier publication from t h i s laboratory a ask ins and Gorz, 1957). In one procedure, designatled method I, o-coumaric acid and total o-hydroxycinnamic acid are determined, and coumarinic acid is estimated by difference; while in the other procedure, method 11, coumarinic acid and total 2-hydroxycinnamic acid are determined, and o-coumaric acid levels are calculated by difference. Both methods depend upon the ultraviolet- mediated interconversion of the non-fluorescent cis isomer and the fluorescent trans form illustrated in Fig. 1. In addition, method II depends upon the fact that o-hydroxycinmmic acid occurs in sweetclover almost exclusively in the form of glucosides (Rudorf and Schwarze, 1958; Kosuge, 19612; Haskins and Gorz, 1961), and sweetclover β- glucosidase readily hydrolyzes coumarinyl glucosl.de but is virtually inert toward o-coumarin glucoside (Kosuge and Conn, 1961).