Complexing of riboflavin with the enolate form of barbituric acid

Riboflavin and the enolate form of barbituric acid form a complex at pH 7.80 as demonstrated by the appearance of two new absorption bands at 400 and 490 nm. The association constant for the complex determined from a Benesi-Hildebrand analysis of absorbances at 490 nm is 30 M^-1. The K evaluated from fluorescence quenching at various barbiturate: riboflavin ratios is 33 M^-1. The complexing of the barbiturate and riboflavin is accompanied by an inhibition of the photo-oxidation of sarcosine to formaldehyde. Under the same conditions, the barbiturate enhances the rate of photo-oxidation of NADH. The 5,5-disubstituted barbiturates do not give a new absorption band with riboflavin and do not inhibit the photo-oxidation of sarcosine, indicating that an enolate configuration of the β-diketone system is involved in complexing with riboflavin.