Diaphorase fluorescence

NADH produced can also be reacted with resazurin in the presence of diaphorase to form resorufin, a highly fluorogenic compound. The fluorescence production is measured at 575 nm and 590 nm. The optimal conditions as well as the sensitivity and linear range of these methods will also be described. 

Recently, a universal enzyme-coupled fluorescence assay for glycosyl transferases was developed. This method is extremely cost-effective and is based on the quantification of nucleotides produced in the glycosyl transfer reaction. The guanosine diphosphate (GDP), uridine diphosphate (UDP), and cytidine monophosphate (CMP) are phos-phorylated with nucleotide kinase in the presence of excess of ATP, generating ADP. Via coupled enzyme reactions involving ADP-hexokinase,glucose-6-phosphate dehydrogenase, and diaphorase, the ADP is utilized for the conversion of resazurin to resorufin, which is then quantified by fluorescence measurement. 

Both the oxidation-reduction potential and the fluorescence of flavin nucleotides are modified profoundly by attachment of the nucleotide to various proteins. Flavin enzymes have been reported to have oxidation-reduction potentials at pH 7 ranging from —0.4 to 0.187. The combination to proteins also results in shifts of the absorption maxima. The 450 m u band is found at 451 mju in Straub s diaphorase and at 455 m/t in Haas yellow enzyme, while the 375 m/t band appears at 359 m/t and 377 m/t in these preparations. Most flavin enzymes do not fluoresce, and it is assumed that the quenching of fluorescence implies binding of the flavin to the enzyme through N-3. Straub s diaphorase, unlike most other flavoproteins, does fluoresce. This may be evidence that this diaphorase is a partially degraded cytochrome reductase. 

The sensitivity was increasec s geral times by the use of fluorimetric determination of NADH. However the preliminary extrac on of BA from serum was still required. In 1976 Mashige et al. reported the first direct enzymatic-fluorimetric assay, based upon the use of the 3 ci -HSD in combination with a resazuri-ne-diaphorase system. The hydrogen of the generated NADH is transferred by diaphorase to resazurine, to yeld the fluorophore, resorfin. The fluorescence of resorfin is then measured at 580 nm with excitation at 560 nm. This method introduced a significant improvement in sensitivity (0.5 mol/l) and clinical usefulness, since no preliminary extraction was required. 

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