3.Detection of proteins by biuret reaction (quantitative determination)

Question 1

The principle of the quantitative determination of proteins is based on…

Answer 1

the determination of the number of peptide bonds, or the quantitation of one constituent amino acid.

Question 2

the method of Quantitative determination of proteins by biuret reaction can be suitable for what proteins concentration?

Answer 2

between 200-400 mg/l.

Question 3

why does the CuSO4 solution has to be in excess compared to the number of peptide bonds?

Answer 3

because otherwise, the amount of Cu2+ ions limit the amount of complex, that means the intensity
of the produced colour

Question 4

what is the Biuret reagent?

Answer 4

dilute stock solution (20 ml) with the diluent solution (80 ml)

Question 5

what are the materials needed for the Quantitative determination of proteins by biuret reaction?

Answer 5

 Diluted blood plasma (80x dilution)
 Biuret reagent: dilute stock solution (20 ml) with the diluent solution (80 ml)
o Stock solution: dissolve 4.5 g of Seignette-salt (potassium-sodium-tartarate) in 40 ml of NaOH solution (0.2 mol/l), then add 1.5 g of anhydrous CuSO4 and 0.5 g of KI. Make up to 100 ml with 0.2 mol/l NaOH solution. (Store in a dark bottle!)
o Diluent solution: make up 5 g of KI to 1000 ml with 0.2 mol/l NaOH solution. The Biuret reagent is provided on our laboratory course as a ready-to-use solution!
 Water bath at 37 °C
 Spectrophotometer

Question 6

what is the stock solution?

Answer 6

Seignette-salt (potassium-sodium-tartarate) dissolve in NaOH solution

Question 7

how do you make the diluent solution?

Answer 7

KI with NaOH solution

Question 8

in which nm we should read the absorbance of the produced color?

Answer 8

546 nm