Biuret Test

Test to detect the presence of Proteins

1. BIURET TEST

2. PROCEDURES

3. PROCEDURE 2.1 Mix 1 mL of egg white solution and 10 drops 6M NaOH in a test tube Add 1 drop 0.5% CuSO 4 solution. Mix well and Record your observation.

4. PROCEDURE 2.1 Dissolve 1 tablet of aspartame or ½ sachet of Equal ® in 2 mL water. Add to the resulting solution 10 drops 6M NaOH and 1 drop 0.5% CuSO 4 solution. Mix well and Record your observation.

5. PROCEDURE 2.1

6. PROCEDURE 2.2 Label 1 test tube TP (Total Protein) , then add 3 drops of blood plasma (or serum) and 3 mL of 23% Na 2 SO 4 solution. Mix well. Transfer half of the mixture from the tube TP to the centrifuge tube. Set aside tube TP. Add 1 mL ether to the mixture in the centrifuge tube, mix thoroughly, and stopper it. Centrifuge for 5 mins. Note the color of the precipitate formed at the junction of 2 liquids (interface) .

7. PROCEDURE 2.2 Ether is highly flammable! Keep it away from open flames.

8. PROCEDURE 2.2 Using a very thin pipette, carefully draw out the lower aqueous layer, taking care not to disturb the precipitate at the interface. Wipe the pipette to remove any precipitate that adheres to it. Transfer the contents of the pipette to a test tube and mark it as A (albumin) . For each of the 2 test tubes (TP & A) , add 1 mL of 6M NaOH and 2 drops of 0.5% CuSO 4 solution. Mix well and compare the color intensity in the test tubes. Record your observations.

9. PROCEDURE 2.2

10. STUDY QUESTIONS

11. A. Principle of Biuret Test

12. Biuret test is conveniently used to detect the presence of proteins in biological fluids. When biuret is treated with dilute copper sulfate in alkaline medium, a purple colored compound is formed. It is believed that the color is due to the formation of a copper coordinated complex or chelate complex.

13. B. Roles of reagents in the test

14. Cu2SO4: this provides the Cu (II) ions which form the chelate complex. NaOH : does not participate in the reaction but provides the alkaline medium CuSO4 + 2NaOH → Cu(OH)2 + Na2SO4

15. CuSO4 + 2NaOH → Cu(OH)2 + Na2SO4

16. C. Will all proteins give a positive biuret test? Why? Will all peptides give a positive Biuret Test?

17. Yes because Biuret test is used in detecting the presence of peptide bonds which links proteins . Yes because Biuret test detects peptides C. Will all proteins give a positive biuret test? Why? Will all peptides give a positive Biuret Test?

18. D. Account for the difference in test results between egg white and aspartame. Show the structure of aspartame.

19. The results for egg whites showed a violet color while the results for Equal ® showed a yellow color. This is because egg whites have proteins which have more than 1 peptide bond and Equal ® only has 1 peptide bond.

20. E. Function of reagents in the extraction of blood proteins.

21. Sodium Sulfate is used as the globulin precipitant. Ether is used as the modified method to separate the serum albumin and globulin

22. F. Chemical composition of the precipitate at the interface after centrifugation. Why should it not contaminate the tube marked A?

23. Answer ni Madam

24. G. Account for the difference in color intensity between samples TP and A.

25. Answer ni Madam

26. H. What group in a peptide or a protein accounts for a positive Biuret Test?

27. Amino Group Carboxyl Group

28. I. Importance of Biuret Test in protein hydrolysis

29. It is a key in explaining protein hydrolysis. Serves as a good index for determining the extent of protein hydrolysis.