Flavoprotein tests

Many of the amino acids originally tested by Krebs were racemic mixtures. When naturally occurring L-amino acids became available the oxidase was found to be sterically restricted to the unnatural, D series. [D-serine occurs in worms free and as D-phosphoryl lombricine (Ennor, 1959)]. It could not therefore be the enzyme used in the liver to release NH3 in amino acid metabolism. D-amino acid oxidase was shown by Warburg and Christian (1938) to be a flavoprotein with FAD as its prosthetic group. A few years later Green found an L-amino acid oxidase in liver. It was however limited in its specificity for amino acid substrates and not very active—characteristics which again precluded its central role in deamination. 

Over the years, a wide variety of diverse proteins have been tested for their potential to serve as the SOs of novel CSPs with different application profiles. For example, flavoprotein 216], avidin and conalbumin [217], as well as other proteins like egg-yolk riboflavin binding protein [218], have been immobilized onto silica. However, neither their enantioselectivity profiles nor their performance was significantly better than those of commercially available protein type CSPs. 

Biological systems (e.g., proteins, cells) excited at wavelengths below 500 nm produce considerable autofluorescence that arises mainly from flavins, flavoproteins, NADH, etc. Labels that can be excited at wavelengths above 500 nm are much less susceptible to optical interference from biological chromophores in the test sample. 

Glycine oxidase has been found in the liver or kidney of all animals tested, and is a flavoprotein which can be resolved under acid conditions into a protein and FAD. 

This work was continued by Dickens, who also used a fraction from autolyzed yeast, TPN, and as the autoxidizable hydrogen carrier, phenazine or a flavoprotein system. Dickens isolated various products, one of which appeared to be a mucture of a phosphopentonic acid and a phosphoketohexonic acid, and others analyzed as 5- and 4-carbon phosphate esters. The 5-carbon compound gave a reaction in the Bial FeClj-orcinol test which was characteristic of pentose. In general, the enzyme preparations of this period did not yield simple reaction products, nor were the methods available for the separation and characterization of the small amounts of products which were isolated. For instance, although suggestions were obtained of the presence of pentose in the products, this was not unequivocally demonstrated. 

Next we tested biologically relevant electron acceptors. EGFP oxidative redding was found to occur in the presence of cytochrome c (ref. 15), flavin adenine dinucleotide (FAD, 6), flavin mononucleotide (FMN, 7), the FAD-containing flavoprotein ucose oxidase (which belongs to the same structural class as ubiquitous and abundant enzymes of cell redox homeostasis, such as glutathione reductase and thioredoxin reductase ) and nicodnamide adenine dinucleotide (NAD, 8). So, excited EGFP was able to reduce... 

Then the extra anion now associated with the oxidised cytochrome may be presumed to pass along the cytochrome electron-transport chain or move inwards by diffusion of the cytochrome-anion complex and be released at a point of lower oxidation potential, for instance, where cytochrome b reacts with flavoprotein. For this process to lead to anion uptake it is postulated that the release of the anion occurs in the inside of the diffusion barrier membrane. This hypothesis when first enunciated aroused considerable interest, not only because of Lundegdrdh s personal reputation as a plant physiologist, but because it was clearly capable of experimental test. 

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