Essential Laboratory Techniques

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Practical 5
Determination of the Activation Energy of an Enzyme Catalysed Reaction

Alkaline phosphatase catalyses the hydrolysis of p-nitrophenyl phosphate (a synthetic substrate) at an optimum pH of 10.0 with the liberation of p-nitrophenol.

The substrate is colourless, but the product p-nitrophenol is yellow in alkaline solution, absorbing maximally at 405 nrn. Thus a convenient assay for this enzyme involves monitoring the change in absorbance of the reaction medium at 405 nm.

Exergonic (i.e. energy producing reactions) exhibit a negative free energy change, -G. Sometimes these reactions occur spontaneously, but generally some energy must be supplied to initiate the reaction; in other words an energy barrier exists between the reactants and the products. This “energy barrier" represents the activation energy of a chemical reaction.

In this practical the activation energy of hydrolysis of p-nitrophenyl phosphate will be determined in the presence of the enzyme alkaline phosphatase.

A suitably diluted solution of alkaline phosphatase
10 mM p-nitrophenyl phosphate
150 M p-nitrophenol (150 nmol/ml)
0.1 M bicarbonate buffer pH 10.0
0.05 M NaOH

a) Preparation of a Calibration Curve
In order to be able to relate absorbance readings at 405 nm to nmol p-nitrophenol/ml produced in the reaction mixtures at 25°C and 35°C a calibration curve must first be set up, to cover the range 0 - 150 nmol/ml (0 -150 M).

Prepare a series of 10 dilutions of p-nitrophenol as detailed in the following table, zero the colorimeter using the reagent blank and measure the absorbance of each solution at 405 nm.

Plot absorbance (vertical axis) against concentration of p-nitrophenol (horizontal axis).

Tube No.| Blank| 1| 2| 3| 4| 5| 6| 7| 8| 9| 10| 150 M p-nitrophenol (ml)| 0| 0.1| 0.2| 0.3| 0.4| 0.5| 0.6| 0.7| 0.8| 0.9| 1.0| Bicarbonate buffer (ml)| 1.0| 0.9| 0.8| 0.7| 0.6| 0.5| 0.4| 0.3|...
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