Question

Advantages and disadvantages of using CDNB assay for GST activity?

Answer 1

Glutathione S-Transferase is an enzyme abundantly found in the liver. It catalyzes the conjugation of GSH (Reduced Glutathione) to electrophilic substrates or any xenobiotic substrates and ultimately helps in the process of detoxification.

Assay principle: GST catalyzes the conjugation of L-glutathione to CDNB through the thiol group of the glutathione. GSH, Reduced Glutathione which is a tripeptide with a free thiol(-SH) group and CDNB is 1-chloro-2,4-dinitrobenzene.

GSH + CDNB(substrates) gives (in presence of GST) GS-DNB Conjugate + HCl

The reaction product, GS-DNB Conjugate, absorbs at 340 nm. The rate of increase in the absorption is directly proportional to the GST activity in the sample.

Advantages of GST-CDNB assay:

1. The measurement of activity is less time-consuming.

2. The visibility of the result is very clear and effective.

3. The experiment has very similar results i.e. good reproducibility.

4. No incubation required. (Just the substrates should be stored in ice along with the GST vials)

5. Rapid results are recorded on the spectrophotometer.

6. CDNB is available commercially at affordable prices.

7. The affinity of CDNB varies for each class of GST i.e. it is specific.

8. This assay helps in detection of abnormalities in liver functioning.

Disadvantages of GST-CDNB assay:

1. GSH (one of the substrates for the assay) is very costly and should be used in limited quantities.

2. The substrates should be prepared before 10 mins of the assay and are required in very minute quantities. Therefore, it cannot be stored.

3. CDNB is light-sensitive reagent, should be always kept covered, otherwise dissociated.

4. CDNB is also associated with various health hazards such as skin, eye, and nasal mucosa irritation. The liquid is flammable.

5. Manual work is more involved.

With very few disadvantages that can be made negligible with proper care, GST-CDNB essay is one of the widely used assay in the labs for measuring GST activity.

I hope this answer helps you!

Best Regards!