The enzyme studied in this experiment is alkaline phosphatase (Type VII-T from bovine intestinal mucosa). This alkaline phosphatase is a dimeric zinc-  containing enzyme (MW ~150 kDa) consisting of two equal subunits. It

catalyzes the hydrolysis of phosphate monoesters to produce inorganic

phosphate; an important compound in many cellular functions. Alkaline

phosphatase has also been proved useful outside the cell (on the bench). For example, molecular biologists use alkaline phosphatase to improve cloning     efficiency and immunologists use this enzyme for labelling and generating

colour in enzyme immunoassays.

To monitor the activity of alkaline phosphatase you will be using p-nitrophenyl phosphate (PNPP); a substrate which upon hydrolysis releases a yellow

coloured product called p-nitrophenol/p-nitrophenolate ion. At pH 8, this product absorbs optimally at 410 nm.

The following experiments will allow you to investigate the enzymatic activity of Alkaline Phosphatase while varying the PNPP substrate concentration,

both in the presence or absence of an inhibitor (sodium phosphate). The

reactions will take place in cuvettes and the rate at which the yellow product is formed will be monitored with the use of the Cary60 UV/Vis

spectrophotometer.

For determining the various enzymatic properties of alkaline phosphatase, including the KM and Vmax, your data analysis will include graphical

interpretations of your data.