Abstract
Commercially available preparations of flavin adenine dinucleotide (FAD) have been found to be 94% pure, the remaining 6% being composed of four or five minor contaminants which can be separated from FAD by reverse-phase high-performance liquid chromatography. FAD purified in this manner has been shown to be 100% pure. One of the contaminants has been identified as riboflavin 5′-pyrophosphate (RPP) by spectroscopic and chemical methods of analysis. This compound has been shown to exhibit biological activity as a weak cofactor for two FAD-requiring enzymes. With the apo-protein of porcine d-amino-acid oxidase, values determined for RPP were 8.4 μ m for K m and 0.10 for V max compared to 0.47 μ m and 0.28 (36 U/mg), respectively, for FAD. With fungal glucose apooxidase, values determined for RPP were 474 n m for K m and 0.02 for V max and 45 n m and 0.09 (105 U/mg), respectively, for FAD. RPP can also inhibit FAD biosynthesis. For bovine liver FAD synthetase, a K i value for RPP against FMN was determined to be 9 μ m where K m for FMN was 5.5 μ m. These studies illustrate the value of riboflavin 5′-pyrophosphate as a flavin analog for use in the study of structure/function relationships within certain flavin-dependent enzymes.
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