Abstract
An isotopic shift of the 31P nuclear magnetic resonance due to 18O bonded to phosphorus of 0.0206 ppm has been observed in inorganic orthophosphate and adenine nucleotides. Thus, the separation between the resonances of 31P18O4 and 31P16O4 at 145.7 MHz is 12 Hz and, in a randomized sample containing ∼50% 18O, all five 16O-18O species are resolved and separated from each other by 3 Hz. Not only does this yield the 18O/16O ratio of the phosphate but, more important, the 18O-labeled phosphate in effect can serve as a double label in following phosphate reactions, for oxygen in all cases and for phosphorus, provided the oxygen does not exchange with solvent water. Thus, it becomes possible to follow labeled phosphorus or labeled oxygen continuously as reactions proceed. Rate studies involving (i) phosphorus and (ii) oxygen are illustrated by continuous monitoring of the exchange reactions between (i) the β phosphate of ADP and inorganic phosphate catalyzed by polynucleotide phosphorylase and (ii) inorganic orthophosphate and water catalyzed by yeast inorganic pyrophosphatase. In the ADP—Pi exchange, the Pi (18O4) yielded an α P(16O318O) and a β P(18O4), proving that bond cleavage occurs between the α P and the α-β bridge oxygen. Among the many additional potential uses of this labeling technique and its spectroscopic observation are: (i) different labeling of each phosphate group of ATP, (ii) to follow rate of transfer of 18O from a nonphosphate compound such as a carboxylic acid to a phosphate compound, and (iii) to follow the rate of scrambling (for example, of the β-γ bridge oxygen of ATP to nonbridge β P positions) and simultaneously the rate of exchange of the γ P nonbridge oxygens with solvent water in various ATPase reactions.
Keywords: ADP—Pi exchange, polynucleotide phosphorylase, site of bond cleavage, inorganic pyrophosphatase, oxygen scrambling
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Selected References
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