International University, Vietnam National University – HCMC

Biochemistry

Prelab: EXPERIMENT 2
PROTEIN QUANTIFICATION APPLYING
HARTREE-LOWRY ASSAY

Full name: Nguyen Thi Lan Anh

Student ID: BTFTIU19041
Group: Durian
1. What fruit was bromelain extracted from? What is the main function
of bromelain?
 Bromelain is extracted from pineapple.
 Bromelain is one of the most popular proteases to use for meat tenderizing.
While there have been studies into the medical use of bromelain, "the
majority of them have methodological issues that make it difficult to draw
definite conclusions", as none definitively established efficacy,
recommended dosage, long term safety, or adverse interaction with other
medications.
2. What are crucial factors that can have effects on enzymatic activity?
Enzyme activity can be affected by a variety of factors, such as
temperature, pH, and concentration.

3. Are fixed times important for exploring/studying the enzymatic

activity? Explain why.
Yes, the study of enzyme activity should be performed within a fixed time.
The fixed-time assay measures enzyme concentration in fixed periods of
time.
4. Why is trichloroacetic acid added into tube of Tyrosine and tube of
water immediately right after putting casein solution into the test
tubes while with respect to tube of sample, trichloroacetic acid is
added at the end of the process?

Trichloroacetic acid is added to inhibit the degradation of casein in the tubes

of Tyrosine and water while in the tube of the sample, we want to measure
the enzymatic activity -> adding the trichloroacetic to stop the reaction.

5. Why shouldn’t we add Tyrosine into tube of sample?

Bromelain was extracted and the activity of the enzyme was measured using
tyrosine as standard and expressed in Units/mL of the enzyme. The amount of
tyrosine released is quantifiable. The more tyrosine that is released from
casein, the stronger the activity of the protease. Therefore, if Tyrosine is added
to the test tube of the sample, it is not possible to accurately determine the
activity of the enzyme

6. How can the activity of enzyme be determined in this experiment?

The activity of bromelain enzyme is determined depending on quantity (μg)
of tyrosine produced from casein degradation under catalysis of enzyme in
1mL of solution or 1mg of bromelain mixture for 1 minute.
7. Student A says that when doing this practical, he can prepare the
tubes of tyrosine and tubes of water first and then measure the
optical density in order to save time. Does he says it right or wrong?
Explain why.
He says it wrong because the test tubes need to be prepared at the same time
so that when measuring the optical density, we can compare their differences
8. Student B says that the more he add bromelain, the faster the
catalytic reaction occurs. Does he says it right or wrong? Explain
why.
It is right because the more bromelain in the solution, more chance for
enzyme bound to substrate lead to the faster of catalytic reaction.
9. Student C says that if he can purify and recover the bromelain used
in this practical, he can continue to use it for the catalytic reaction next
time in order to save the enzyme. Does he says it right or wrong?
Explain why.
It is wrong because after the reaction, some bromelain molecules are
inactive so it will affect to next result leading to having the wrong result in
the next experiment.
10. Student D says that there is a paradox in enzymatic activity. He
supposes that protease can degrade itself because it is also a protein.
Does he says it right or wrong? Explain why.
The enzyme works on specific substrates that mostly are simpler chemical
compounds, less complicated than enzyme-type protein complex. So, unless
the enzymatic protein is degraded first and or becoming a less simple
protein that protease will recognize, protease will not do its job to destroy
its own kind.