Product Overview :
HPRT Assay Kit provides an enzymatic tool for continuous spectrophotometric monitoring of HPRT activity in a convenient 96-well plate format. In the assay, HPRT activity is measured as a rate of production of IMP, which is oxidized by recombinant IMPDH enzyme with simultaneous reduction of NAD+ to NADH measurable by absorbance at 340nm.The assay is developed for measuring HPRT activity in vitro or in lysates of cells. For maximal accuracy, the assays with cell lysates are run with and without PRPP in parallel. The absorbance rate observed in the absence of PRPP is used as blank and is subtracted from the absorbance rate measured in the presence of PRPP.
Description :
Hypoxanthine phosphoribosyltransferase is a purine salvage enzyme that catalyzes the reversible transfer of the 5-phosphoribosyl moiety from α-D-5-phosphoribosyl-1-pyrophosphate (PRPP) to a purine base (hypoxanthine or guanine) to form a nucleoside monophosphate (inosine monophosphate or guanosine monophosphate, respectively). In the presence of pyrophosphate, HPRT enzyme catalyzes also the hydrolysis of IMP and GMP, although this reverse reaction is much less favored than forward one.
Human HPRT enzyme does not hydrolyse XMP. HPRT1 gene is one of the best characterized in the human genome for two reasons: (i) HPRT1 gene is widely used as a somatic cell genetic marker in genotoxicity / mutagenicity studies; (ii) the defects within the human enzyme are associated with inherited gouty arthritis and Lesch–Nyhan syndrome and more than 300 disease-associated mutations in human HPRT1 gene leading to partial or complete deficiencies of the HPRT enzyme have been described. In view of the high variability of HPRT1 gene, a rapid biochemical assay would be useful both for basic science and clinical research.
In addition, since most parasitic protozoan are obligate auxotrophs of purines and entirely depend therefore on their purine salvage pathways, protozoan HPRT enzyme is an attractive target for the discovery of new anti-parasitic drugs. The enzymatic microplate assay enabling monitoring of HPRT activity may therefore accelerate the search of new anti-parasitic drugs.
Storage :
Once dissolved, the reagents provided in the kit are not stable and should be stored on ice and used the day of preparation.
Kit Components :
This kit allows performing 24 analysis in a time (8 samples in triplicate, 12 samples in duplicate).
A standard HPRT Assay Kit contains:
one tube "Cofactor 1" (DTT);
one tube “Cofactor 2" (NAD);
one tube "Bacterial IMPDH" ;
one tube "Reaction buffer 10x" (1mL, contains hypoxanthine);
one tube of "Human Recombinant HPRT" for preparing enzyme solution at 75mU/ml(94.6nmol/h/ml);
one transparent 96-well plate.
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