Determination of the Activation Energy of an Enzyme Catalysed Reaction
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)
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)