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Enzymes I: Determination of acid phosphatase activity

Posted on Feb 08 2013 by Tracey Kuit

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Experiment Overview

This experiment looks at the area of enzyme kinetics using an acid phosphatase from potato. Students measure the breakdown of p-nitrophenyl phosphate by phosphatase, by measuring one of the products, p-nitrophenol (PNP). PNP can be measured spectrophotometrically as it is coloured (in alkaline solution). The other product is inorganic phosphate.


              substrate                       enzyme                             products

     p-nitrophenyl phosphate                    p-nitrophenol   +  inorganic phosphate

                                              phosphatase           (yellow)


In the first part of the experiment students prepare a series of standard solutions containing different amounts of PNP and measure their extinction at 400 nm in the spectrophotometer.  Students graph the extinction values against the amount of p-nitrophenol and later use this to determine the amounts of p-nitrophenol released by enzyme action.

In the second part of the experiment students prepare an enzymatic reaction by mixing the enzyme and buffer with the p-nitrophenyl phosphate substrate.   At intervals after the start of the reaction, students remove aliquots of the reaction mixture and add them in turn to each of the tubes containing NaOH. The NaOH will stop the reaction, and students then determine the amount of coloured p-nitrophenol product released as a function of time by measuring the extinction of the contents of these tubes in a spectrophotometer and interpolating the results from the standard curve.


Students finally graph the amount of p-nitrophenol produced in the reaction against time and use this graph to calculate the initial rate of the reaction, and then calculate the specific activity of the enzyme.


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