1) Substitute the solution in the video into the equation below.

(2 marks)
E  +   S   ⇔  E-S  →  P

Protease + Casein ⇔ Protease-Casein complex Amino acid tyrosine

 
2) State the function of the TCA and Folin & Ciocalteau’s in the video. (2 marks)

The solution will react to tyrosine to generate measurable color change that will be directly
related to the activity of protease

3) Define colorimetric assay. (1 mark)

Colorimetric analysis is a method of determining the concentration of a chemical element or

chemical compound in a solution with the aid of a color reagent. It is applicable to both
organic compounds and inorganic compounds and may be used with or without an enzymatic
stage.
 
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
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)

 0.8

0.7 f(x) = 0.9 x + 0.02

R² = 1
0.6

0.5
405nm

0.4

0.3

0.2

0.1

0
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9
Choline (μmol)

b) Determine the activity of the enzyme if the absorbance of enzymatic activity gave
a reading of  0.552 (4 marks).

Since y = 0.9014x + 0.0173, substitute y=0.552

0.552 = 0.9014x + 0.0173
X = 0.5932 (µmol choline equivalent released)

choline equivalent relese ( μml ) x total∈assay(ml)

Activity=
time∈assay ¿¿

0.5932 μml x 7.0 ml

=
10 min x 1 ml x 1 ml

units
= 0.4152 ml
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)

Units/mL enzyme
Activity = mg solid /mL enzyme

units
0.4152
ml
= mg x5
10
ml

= 0.2076 units/mg