jlias sudbioa
Practical 5
Determination of the Activation
Energy of an Enzyme Catalysed
Reaction
Prof Declan Naughton
PR MB 1074 Office Hours Friday 1‐3 pm
(I) Introduction
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. http://www.gbiosciences.com/ResearchProducts/PhosphataseAssay‐desc.aspx
Aim: To determine the activation energy of the enzyme alkaline phosphatase
(II) Method
a) Preparation of a Calibration Curve
b) Determination of Activation Energy of the Reaction
4
Preparation of a Calibration Curve
Plot absorbance (vertical axis) against concentration of p‐nitrophenol (horizontal axis).
Determination of Activation Energy of the Reaction
Determination of Activation Energy of the Reaction
Add 0.6 mL of enzyme solution to the reaction tubes and mix
Determination of Activation Energy of the Reaction
Add 0.6 mL of enzyme solution to the reaction tubes and mix
Take 1 mL samples at given time points & add to tubes containing 2 mL of 0.05 M NaOH
Measure absorbance against calibration blank from the calibration curve.
Planning
1. Turn on spectrometer (and wait to internally calibrate)
2. Zero spectrometer with blank from calibration curve (keep this sample for further runs)
3. Work in teams of 3
4. 1 student on calibration curve
5. 1 student on 25 oC run
6. 1 studnt on 35 oC run.
(III) Treatment of Results
A) Convert your absorbance readings to nmol p‐nitrophenol/mL.
Treatment of Results
A) Convert your absorbance readings to nmol p‐nitrophenol/mL.
B) Draw a progress curve of nmol p‐nitrophenol/mL against time for each temperature.
Treatment of Results
A) Convert your absorbance readings to nmol p‐nitrophenol/mL.
B) Draw a progress curve of nmol p‐nitrophenol/mL against time for each temperature.
C) Calculate the initial rate of reaction (V1 & V2) at each temperature as
nmol
Determination of the Activation
Energy of an Enzyme Catalysed
Reaction
Prof Declan Naughton
PR MB 1074 Office Hours Friday 1‐3 pm
(I) Introduction
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. http://www.gbiosciences.com/ResearchProducts/PhosphataseAssay‐desc.aspx
Aim: To determine the activation energy of the enzyme alkaline phosphatase
(II) Method
a) Preparation of a Calibration Curve
b) Determination of Activation Energy of the Reaction
4
Preparation of a Calibration Curve
Plot absorbance (vertical axis) against concentration of p‐nitrophenol (horizontal axis).
Determination of Activation Energy of the Reaction
Determination of Activation Energy of the Reaction
Add 0.6 mL of enzyme solution to the reaction tubes and mix
Determination of Activation Energy of the Reaction
Add 0.6 mL of enzyme solution to the reaction tubes and mix
Take 1 mL samples at given time points & add to tubes containing 2 mL of 0.05 M NaOH
Measure absorbance against calibration blank from the calibration curve.
Planning
1. Turn on spectrometer (and wait to internally calibrate)
2. Zero spectrometer with blank from calibration curve (keep this sample for further runs)
3. Work in teams of 3
4. 1 student on calibration curve
5. 1 student on 25 oC run
6. 1 studnt on 35 oC run.
(III) Treatment of Results
A) Convert your absorbance readings to nmol p‐nitrophenol/mL.
Treatment of Results
A) Convert your absorbance readings to nmol p‐nitrophenol/mL.
B) Draw a progress curve of nmol p‐nitrophenol/mL against time for each temperature.
Treatment of Results
A) Convert your absorbance readings to nmol p‐nitrophenol/mL.
B) Draw a progress curve of nmol p‐nitrophenol/mL against time for each temperature.
C) Calculate the initial rate of reaction (V1 & V2) at each temperature as
nmol