General description

Purine Nucleoside Phosphorylase (PNP) (E.C. 2.4.2.1.) is an enzyme involved in purine metabolism and it catalyzes the cleavage of the glycosidic bond of ribo- or deoxyribonucleosides, in the presence of inorganic phosphate as a second substrate, to generate the purine base and ribose-1-phosphate or deoxyribose-1-phosphate. It is one of the enzymes of the nucleotide salvage pathways that allows the cell to produce nucleotide monophosphates when the de novo synthesis pathway has been interrupted or is non-existent (as is the case in the brain). PNP deficiency leads to severe combined immunodeficiency and also leads to hypouricemia and reduced urinary excretion of uric acid.

Principle

In this Purine Nucleoside Phosphorylase Activity Assay, hypoxanthine formed from the breakdown of inosine is detected via a multi-step reaction, resulting in the generation of an intermediate that reacts with the PNP Probe. The fluorescent product is measured at λex = 535nm/λem = 587nm. Limit of quantification is 0.005 µU of recombinant purine nucleoside phosphorylase.

Suitability

Suitable for the detection of Purine Nucleoside Phosphorylase activity in cell and tissue lysate or purified recombinant protein