Question

Amino Acids and Proteins Lab, post lab questions

1.Which samples gave positive results in the biuret test? Were there any differences in the color and intensity of the positive test results? How general is the biuret test for detecting proteins of different types?

2. Which amino acids are identified by means of the xanthoproteic test? Which protein samples gave positive xanthoproteic test results? Comment on the composition of the protein samples based on the results of this test.

3. Which amino acids are identified by means of the Sakaguchi test? Which protein samples gave positive Sakaguchi test results? Comment on the composition of the protein samples based on the results of this test.

4. Which amino acids are identified by means of the nitroprusside test? Which protein samples gave positive nitroprusside test results? Comment on the composition of the protein samples based on the results of this test.

5. Compare and contrast the effect of strong acid (HCl) on albumin, casein, and gelatin. Which protein was most sensitive to the action of strong acid? Least sensitive?

6. Which metal salts (CuSO4 and AgNO3) caused albumin denaturation? How does this observation relate to the toxicity of silver salts versus copper salts?

7. You have just been to the doctor’s office to receive an inoculation. Before administering the injection, the doctor wipes the area with an alcohol swab. Do the results for albumin denaturation support the use of isopropyl alcohol as a disinfectant? Explain.

8. The biuret test is used to identify proteins. Compare the results obtained in the biuret test with albumin and the filtrate after the salting-out procedure in Part B. How effective is the “salting-out” procedure with ammonium sulfate?

9. Is denaturation of albumin by ammonium sulfate reversible or irreversible? Explain on the basis of your observations for the biuret test with albumin and the redissolved precipitate, respectively.

Answer 1

Q1-Biuret test is used to detect presence of peptide bonds which is characteristic of proteins(-CO-NH-) linkage. Presence of protein using biuret test is determined by treating protein with copper salt in presence of strong base and detection is confirmed by formation of purple colour of varying strength.Greater the number of peptide linkages present greater will be the intensity of color after biuret test and hence shift in absorption bands occur.

Q2-Amino acids like throwing and tryptophan can be detected using xantoprotic test as this is meant for detection of proteins with aromatic rings by treating those corresponding proteins with concern nitric acid. A presence of protein is detected by yellow ring or Ppt.

Q3- This test is used for detecting gaunidine group in proteins like arginine. Sakaguchi reagent is solution of alpha napthol with sodium hypobromite and gaunidine group of arginine protein reacts with this reagent giving red colored product detecting presence of protein for e.g. arginine glutamate

Q4- Nitroprusside test is used to detect proteins with free think groups like cystine(-SH). Protein is treated with sodium or potassium Nitroprusside in presence of weak base like ammonium hydroxide giving red color showing presence of free thiol groups.

Q5-Albumin and casein on treating with strong acid gives white coagulate while gelatin gives colorless coagulate. Off this proteins, albumin is more sensitive while gelatin is least sensitive.

Q6- Silver nitrate is acidic salt andso is copper sulphate but silver nitrate denatures protein by providing H+ as These ions break hydrogen bonds thereby maintaining original structure of proteins. While copper sulphate has tendency to make water enter the core of protein and cause disruption of bonds with protein molecule. It is capable of breaking disulphide customer bonds bonded to sulphydryl groups,this interaction is little toxic as it changes structure of protein.

Q7- Alcohol applied on skin is basically meant to enter bacterial cell wall of skin and enter cell wall and albumin of skin and denature albumin protein and enzymes in cell. Alcohol denatures protein by entering or interrupting side chain intramolecular hydrogen bonding.

Q8-Albumin gives positive protein biuret test forming purple color solution. Ammonium sulphate is salt of weak base and strong acid. Due to good acidic strength, as concentration of salt increases, solubility of protein decreases and at a very higher degree o f ionic strength, protein gets precipices out and thus salting out occurs.

Q9-Degree of reversibility of denaturation of albumin by using ammonium sulphate depends on pH of medium. Till pH is maintained, protein is stable or else the precipitation of protein turns out to be reversible