When you ran the ALP assay using pNpp, you could use a high concentration of pNpp....
When you ran the ALP assay using pNpp, you could use a high concentration of pNpp. If you run the ALP assay using pNp and ATP, you must use a lower concentration of pNp. Why?
Homework Answers
Answer:
ALP will catalyze a hydrolysis reaction separation of the phosphate group and resulting in the formation of products p-nitrophenol (PNP) and hydrogen phosphate. p-nitrophenol absorbs light at 405 nm so we can measure change in p-nitrophenol as ALP converts PNPP into PNP and hydrogen phosphate.
p-nitrophenolphosphate Alkaline Phosphatase > p-nitrophenol + H2PO4-
ALP activity ( U/ml) = A/V/T
A= amount of pNP generated by sample
V= volume of sample added in the assay
T= time
ALP assy using pNpp, we use high concentration of pNpp and similarly, ALP assay using pNp and ATP, we use lower concentration of pNp because of alkaline phosphatase enzyme dephosphatase substrate so less absorbs we observed. Hence use of ATP to pNp which increase amount of absorbs so that amount of enzyme activity measured.
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