jlias sudbioa Essay

Practical 5

Determination of the Activation�

Energy of an Enzyme Catalysed

Effect

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

four. 1  student on calibration curve

your five. 1  student on 25 oC work

6. 1 studnt on 35 oC work.

(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 p‐nitrophenol/min/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. C) Calculate the initial rate of reaction (V1 &  V2) at each temperature as� nmol p‐nitrophenol/min/mL.

D) Calculate the activation energy for the enzymatic hydrolysis of p‐nitrophenol phosphate using the expression given.

R = the gas constant,  8. 314 J/mol/°K,  T = temperature °K� [K] = [°C] + 273.15

Final report

1 . Give your full name,  group,  KU number and practical title &  number. 2 . Tabulate the results used to make the calibration curve.  (T1) three or more. Tabulate the absorbance readings before and after conversion to nmol p‐nitrophenol/mL� (III) a (T2)

4. Tabulate results from the progress curve (III) b (T3) your five. Give the calculation and results for the initial rate of reaction (III) c (T4 &  result) 6. Give the calculation and result for the activation energy calculation (III) d 7. Briefly comment on your result (max 100 words).  (Discussion) eight. Deadline is next Monday at 9 am. �

General sensible timetable: Wednesday TB2

Week

1

9-10

10-12

1-3

4-6

Guide A-C

Practical 3* C

Practical 3* A

Sensible 3* B

(*Wed 12-1)

(*Wed 1-2)

(*Wed 2-3)

2

Guide D-F

Sensible 3* N

Practical 3* D

Functional 3* At the

(*Wed 12-1)

(*Wed 1-2)

(*Wed 2-3)

3

Guide G-I

Functional 3* I

Practical 3* G

Useful 3* H

(*Wed 12-1)

(*Wed 1-2)

(*Wed 2-3)

4

your five

6

several

8

being unfaithful

10

eleven

Tutorial A-C

Practical 5 A

Useful 4 W

Practical 4 C

Guide D-F

Functional 4 Deb Practical 4 E

Functional 4 Farreneheit

Tutorial G-I

Practical four G Useful 4 They would Practical some I

Article A-C

Functional 5 M

Practical five C Practical 5 A

Tutorial D-F

Practical a few E

Useful 5 N

Tutorial G-I

Practical your five H Useful 5 I actually

Practical 5 D

Sensible 5 G...



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