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Lab Report 3 (Bio462)

This document provides instructions for an experiment to determine enzyme activity. Students are asked to: 1) Substitute components in an enzymatic reaction equation. 2) Explain the purpose of Folin & Ciocalteau's reagents in detecting tyrosine. 3) Use a standard curve to calculate the activity and specific activity of acetylcholinesterase from absorbance data, accounting for dilution during sample preparation. The experiment measures enzymatic conversion of substrates to products that are detected colorimetrically.

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Allisya Nasir
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149 views

Lab Report 3 (Bio462)

This document provides instructions for an experiment to determine enzyme activity. Students are asked to: 1) Substitute components in an enzymatic reaction equation. 2) Explain the purpose of Folin & Ciocalteau's reagents in detecting tyrosine. 3) Use a standard curve to calculate the activity and specific activity of acetylcholinesterase from absorbance data, accounting for dilution during sample preparation. The experiment measures enzymatic conversion of substrates to products that are detected colorimetrically.

Uploaded by

Allisya Nasir
Copyright
© © All Rights Reserved
Available Formats
Download as PDF, TXT or read online on Scribd
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UNIVERSITI TEKNOLOGI MARA

CAWANGAN NEGERI SEMBILAN, KAMPUS KUALA


PILAH

BIO462
BIOCHEMISTRY

LABORATORY PRACTICAL 3

SEMESTER MARCH - AUGUST 2022 (ODL)

Name
Student ID
Group
EXPERIMENT 3: DETERMINATION OF ENZYME ACTIVITY

1. Substitute the solution in the video into the equation below. (1 mark)
E+S→ E-S → P
Protease + Casein → Protease – Casein complex → Tyrosine

2. State the function of the Folin & Ciocalteau’s in the video. (1 marks)
Folin and Ciocalteau's function as reagents in a solution that reacts with tyrosine to
produce a visible colour change (blue colour) that is related to protease activity.

3. 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
0.067 0.05
0.113 0.1
0.197 0.2
0.395 0.4
0.729 0.8

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


b) Determine the activity of the enzyme if the absorbance of enzymatic activity
gave a reading of 0.552 (3 marks).
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. (2
marks)

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