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Lab Report Biochemistry (BIO 462)

This lab report describes an experiment to determine enzyme activity through a colorimetric assay. The experiment involves reacting acetylcholinesterase enzyme with acetylthiocholine iodide substrate, then adding reagents to produce a blue chromophore. Absorbance readings are used to construct a standard curve relating chromophore concentration to choline concentration. This allows calculating the activity of acetylcholinesterase based on an absorbance reading of 0.552, found to be 5.7394 micromoles of choline produced. Accounting for a 5x dilution of the enzyme, the specific activity is calculated to be 2.8697 units per milligram of protein.

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Iman Farha
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1K views3 pages

Lab Report Biochemistry (BIO 462)

This lab report describes an experiment to determine enzyme activity through a colorimetric assay. The experiment involves reacting acetylcholinesterase enzyme with acetylthiocholine iodide substrate, then adding reagents to produce a blue chromophore. Absorbance readings are used to construct a standard curve relating chromophore concentration to choline concentration. This allows calculating the activity of acetylcholinesterase based on an absorbance reading of 0.552, found to be 5.7394 micromoles of choline produced. Accounting for a 5x dilution of the enzyme, the specific activity is calculated to be 2.8697 units per milligram of protein.

Uploaded by

Iman Farha
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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LAB REPORT BIOCHEMISTRY

(BIO 462)

EXPERIMENT 4: DETERMINATION OF
ENZYMES ACTIVITY

NAME IMAN FARHA HANISAH BINTI DANIEL HASNI


STUDENT NUMBER 2019702307
GROUP AS2464B
LECTURER’S NAME DR NUR NADIAH MD YUSOF
DATE OF EXPERIMENT 27TH APRIL 2020
Determination of Enzymes Activity
1. Substitute the solution in the video into the equation below. (2 marks)
E + S → E-S→ P
Protease + Casein → Tyrosine → Chromophore

2. State the function of the TCA and Folin & Ciocalteau’s in the video. (2 marks)
TCA – To stop enzyme reaction and denature casein
Folin & Ciocalteu – react with tyrosine to generate blue colored chromophore.

3. Define colorimetric assay. (1 mark)


A technique used to determine the concentration of coloured compound in a solution.

4. In an enzymatic reaction, 1.0 mL of acethylcholinesterase was reacted with 2.0 mL


of acethylthiocholine iodide. 2.0mL of phosphate buffer was then been added in.
Incubation for 10 minutes took place before 1.0 mL of acetic acid and 1.0 mL of
dithiobisnitrobenzoic acid was added up to the mixture. The mixture was further
incubated for 10 minutes and 1.0 ml of the sample was placed in cuvette for
absorbance reading at 405 nm.

Using the data for the product standards below,

405 nm Choline (μmol)

0.00 0.05

0.067 0.1

0.113 0.2

0.197 0.3

0.395 0.4

0.729 0.8
a) Plot a standard curve based on the data given. (3 marks)

450 nm vs Choline (μmol)


0.8
0.729
0.7
0.6
y = 0.1347x - 0.2211
0.5
0.4 0.395
450 nm

0.3
0.2 0.197
0.1 0.113
0.067
0 0
0.05 0.1 0.2 0.3 0.4 0.8
-0.1
-0.2

Choline (μmol)

b) Determine the activity of the enzyme if the absorbance of enzymatic activity


gave a reading of 0.552 (4 marks).
Y = 0.1347x – 0.2211
0.552 = 0.1347x – 0.2211
0.1347x = 0.7731
X = 5.7394
c) Calculate the specific enzyme activity if the total protein content is 10 mg/mL
and the enzyme has been diluted 5X before enzymatic reaction takes place. (3
marks)
Total activity = specific activity x total protein content

5.7394 = specific activity x 10

Specific activity = 0.5739

Diluted enzyme = 0.5739 x 5 = 2.8697

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