Abstract
8-Azidoflavins have been synthesized and their potential as photoaffinity labels for flavoproteins has been explored. They are very photolabile, and in aqueous media they react with solvent to yield 8-aminoflavins and 8-hydroxlaminoflavins as the main products. They fulfill the criteria expected of a good photoaffinity label, since they bind stoichiometrically at the flavin-binding site of flavoproteins, thus minimizing problems of nonspecific labeling. Second, they absorb strongly in the visible, so that the reactive nitrene can be generated without short wavelength light, minimizing the possibility of light-induced damage of the protein. Third, in the absence of light, 8-N3-flavins are stable, permitting a study of their binding to apoproteins. 8-Azidoflavins have been bound to hen egg white riboflavin-binding protein, Megasphera elsdenii flavodoxin, yeast Old Yellow Enzyme, Aspergillus niger, glucose oxidase, and pig kidney D-amino acid oxidase, and the effect of exposure to visible light has been determined. Only small extents of covalent attachment of the flavin to the protein were found with flavodoxin, D-amino acid oxidase, and Old Yellow Enzyme; much more extensive labeling was obtained with glucose oxidase and riboflavin-binding protein. In addition to their photoreactivity, 8-azidoflavins have been found to be converted to 8-aminoflavins by reaction with sulfite or upon reduction. Similar reactions occur with 8-hydroxylamino-, 8-(O-methyl)hydroxylamino-, and 8-hydrazinoflavins, which serve as models for possible flavin-protein covalent linkages which could be formed in the photolabeling procedure. Some of the properties of these flavins, which were obtained by reaction of 8-F-flavin with the corresponding nucleophiles, are also described.
Highlights
From the $Departmentof Biological Chemistry, The University of Michigan, A n n Arbor, Michigan 48109 and the VFakultat Biologie der Uniuersitat, 0-7750 Konstanz, Federal Republic of Germany
In thaebsence of light, 8N3-flavins are stable, permitting a studoyf their binding toapoproteins. 8-Azidoflavins have been bound to hen egg white riboflavin-binding proteinM, egasphera elsdenii flavodoxin, yeast Old Yellow Enzyme, Aspergillus niger, glucose oxidase, and pig kidney D-amino toaffinity reagents can be used to identify the location of the binding site within the protein, even when such sites do not contain highly reactive nucleophiles
The spectral changes seen upon irradiation of 8-N3-FAD enzymewere similar to those seen with riboflavin-binding protein and free 8-N3-riboflavin, but the decrease in absorbance was less (Fig. 8).The light sensitivity of 8-N3-FAD-~amino acid oxidase is similar to that of free 8-N3-riboflavin, in contrast towhen bound to riboflavin-binding protein
Summary
From the $Departmentof Biological Chemistry, The University of Michigan, A n n Arbor, Michigan 48109 and the VFakultat Biologie der Uniuersitat, 0-7750 Konstanz, Federal Republic of Germany. This was taken advantage of to pass a sample of riboflavin-binding protein through three cycles of labeling followed bydialysis to remove noncovalently bound flavin, in order to increase the amount of covalently bound flavin to approximately 30%.
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