Polynucleotide phosphorylase-based photometric assay for inorganic phosphate.
Polynucleotide phosphorylase is a prokaryotic enzyme that catalyzes phosphorolysis of polynucleotides with release of nucleotide diphosphates. By taking advantage of this property, we developed a photometric assay for inorganic phosphate. In the presence of polyadenylic acid, phosphate is converted into adenosine 5'-diphosphate (ADP) by this enzyme. ADP then reacts with phosphoenolpyruvate in a pyruvate kinase-catalyzed reaction, thus giving rise to adenosine 5'-triphosphate and pyruvate. Finally, pyruvate oxidizes reduced nicotinamide adenine dinucleotide (NADH) through the action of L-lactate dehydrogenase, with concomitant decrease in absorbance at 340 nm. As expected, in this detection system 1 mol of NADH was oxidized per mole of phosphate. The assay showed an excellent reproducibility, as the standard deviations never exceeded 5%. It also was shown to be unaffected by several compounds that are regarded as major interferents of the traditional colorimetric assays. Absence of interference was also demonstrated when determining phosphate content in different biological samples, such as human serum and perchloric acid extracts from Escherichia coli, yeast, and bovine liver. An E. coli strain overexpressing His-tagged polynucleotide phosphorylase developed in our laboratories allowed quick and straightforward purification of enzyme, making the assay feasible and convenient. Since all other reagents required are inexpensive, the assay represents a cheaper alternative to commercially available phosphate assay kits.
Ghetta
Andrea
A
Dipartimento di Biotecnologie e Bioscienze, Università di Milano-Bicocca, Piazza della Scienza 2, I-20126 Milan, Italy.
Matus-Ortega
Maura
M
García-Mena
Jaime
J
Dehò
Gianni
G
Tortora
Paolo
P
Regonesi
Maria Elena
ME
D000244:Adenosine Diphosphate / Q000032:analysis; D004926:Escherichia coli / Q000201:enzymology; D010710:Phosphates / Q000032:analysis*; D010783:Photometry / Q000379:methods*; D011117:Polyribonucleotide Nucleotidyltransferase / Q000302:isolation & purification / Q000378:metabolism*
D010710:Phosphates; D000244:Adenosine Diphosphate; D011117:Polyribonucleotide Nucleotidyltransferase