Determination of the Activation Energy of an Enzyme Catalysed Reaction

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INTRODUCTION
Alkaline phosphatase catalyses the hudrolysis 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. Sometimes these reactions occur spontaneously, but generally some energy must be supplied to initiate the reaction; in other word an energy barrier exists between the reactants and the products. The “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.

REAGENTS
* A suitably diluted solution of alkaline phosphatase
* 10 nM p-nitrophenyl phosphatase
* 150  μM p-nitrophenyl phosphate
* 0.1 M bicarbonate buffer pH 10.0
* 0.05 M NAOH

Method
a) PREPARATION OF 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 OC and 35 oC a calibration curve must be set up, to cover the range 0-150 nmol/ml (0-150µM)

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

TUBE NO.| BLANK| 1| 2| 3| 4| 5| 6| 7| 8| 9| 10| 150 µM p-nitrophenol| 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| 0.2| 0.1| 0| NAOH (ml)| 2.0| 2.0| 2.0| 2.0| 2.0| 2.0| 2.0| 2.0| 2.0| 2.0| 2.0| Concentration (nmol/ml)| 0| 5| 3| 4.5| 6| | | | | | | ABSORPTION (A)|...
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