Agronomy and Horticulture Department


Date of this Version



Published in BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Vol. 6, No. 4,1961. Copyright 1961. Used by permission.


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 but is virtually inert toward o-coumarin glucoside (Kosuge and Conn, 1961).