FACULTY OF APPLIED SCIENCE

BACHELOR OF SCIENCE (HONOURS) BIOLOGY

BIO462
EXPERIMENT 3: DETERMINATION OF ENZYMES
ACTIVITY
NAME: AIDA NABILAH BINTI ABDUL HADI
STUDENT ID: 2021524367
CLASS: AS2012B1
LECTURER’S NAME: MOHD SYAHRIL MOHD ZAN
SEMESTER OCT 2021 FEB 2022
EXPERIMENT 3: DETERMINATION OF ENZYMES ACTIVITY
Answer all the questions.

1. Substitute the solution in the video into the equation below. (1 mark)
Protease + Casein → Protease-casein → Tyrosine

2. State the function of the Folin& Ciocalteau’s in the video. (1 mark)

The function of the Folin& Ciocalteau’s in the video is the solution will react with tyrosine to
generate a measurable color change which directly related to the activity of proteases.

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 products 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)

a) Determine the activity of the enzyme if the absorbance of enzymatic activity gave a
reading of 0.552 (3 marks).
y = mx + c y = absorbance
y = 0.9014x + 0.0173 x = choline

y = 0.9014x + 0.0173
0.552 = 0.9014x + 0.0173
x = 0.6 µmol

Activity of enzyme = choline released x total assay

time in assay x volume of enzyme x volume in cuvette
= 0.6 µmol x 7.0 mL
10 min x 1.0 mL x 1.0 mL
= 0.42 units/mL
b) 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)

Specific enzyme activity = 0.42 units/mL

10 mg/mL ÷ 5
= 0.2 units/mg