Spectrophotometric determination of fructose-1:6-diphosphate, hexosemonophosphates, adenosinetriphosphate and adenosinediphosphate

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Abstract
An enzymic method for the detn. of phosphorylated sugars and energy-rich compounds is descr. The method depends upon the enzymic conversion of these compounds to dihydroxyacetonephosphate, which then reacts with reduced diphosphopyridine nucleotide (DPN) in the presence of glycerolphosphate dehydrogenase. The amt. of reduced DPN reacting is determined spectrophoto-metrically. The method is highly sensitive, 0.05 [mu] mole of phosphorylated sugar or energy-rich phosphate being measured with an accuracy of a few %. In a complex mixture separate analyses are obtained for (a) hexosediphosphate + triosephosphates, (b) hexosemonophosphates (glucose-6-phosphate, glucose-1-phosphate, fructose-6-phosphate, but not fructose-1-phosphate), (c) adenosine triphosphate and (d) other energy-rich compounds (adenosine diphosphate, creatine-phosphate, phosphopyruvate (which is over-estimated by 50%). The only substances which interfere are (i) pyruvate and oxaloacetate, each molecule of which reacts as half a molecule of hexosediphosphate and (ii) phosphoglycerate, which behaves like phosphopyruvate. Pyruvate and oxaloacetate may be separately determined with lactic and malic dehydrogenases. Analyses of prepns. of phosphorylated sugars and adenine nucleotides, either prepared in the laboratory or obtained commercially, were made both by the new method and by conventional chemical methods. Agreement was very close in most cases. Where there was disagreement, this was traced to the presence in the prepns. of impurities which are estimated by the chemical but not by the enzymic method.