RATES OF INTERACTIONS OF SUPEROXIDE WITH VITAMIN-E, VITAMIN-C AND RELATED-COMPOUNDS AS MEASURED BY CHEMILUMINESCENCE

The rate constants for the interactions of superoxide with vitamin E (alpha-tocopherol), vitamin C (ascorbic acid) and their related compounds have been measured by a chemiluminescence method. A strong chemiluminescence of a constant intensity was observed when xanthine oxidase was added to an aqueous solution of hypoxanthine and a Cypridina luciferin analog, 2-methyl-6-phenyl-3-7-dihydroimidazo[1,2-a]pyrazin-3-one (CLA). Vitamin E, vitamin C and their related compounds competed with CLA to react with superoxide and reduced the chemiluminescence intensity. From a kinetic analysis of the effect of addition of these compounds on the chemiluminescence intensity, the rate constants for their interactions with superoxide were measured at 25-degrees-C and pH 7.8. The rate constants were obtained as 3.3 . 10(5) and 1.7 . 10(4) M-1 s-1 for ascorbate and 2-carboxy-2,5,7,8-tetramethyl-6-chromanol, respectively, and also as 4.9 . 10(3) and 4.5 . 10(3) M-1 s-1 for alpha-tocopherol incorporated into soybean and dimyristoyl phosphatidylcholine liposomal membranes, respectively. It has been shown that this method is a sensitive and a quick method which can be applied for measurement of the reactivities of various natural and synthetic compounds toward superoxide. In addition it has been shown that this method can also be applied to the heterogeneous system as well as homogeneous solution, which makes it more versatile and useful for the study in biochemistry.