Question

Proteins are composed of amino acid units bound by peptide bonds. By the addition of the enzyme Bacterial Protease these bonds are broken and the individual amino acids are freed. A simple test for free amino acids is the use of the reagent Ninhydrin which turns purple in the presence of amino acids but remains yellowish in the presence of protein.

I. Label one 250 ml beaker A and one 250 ml beaker B. To each beaker add l 00 ml of water and one half of the curd. Stir well to break up the curd.

2. To beaker A add 1.0 g of the enzyme Bacterial Protease. Stir well and allow standing 5 minutes. Add no enzyme to beaker B. Stir both beakers well and allow to stand 5 minutes.

3. After 5 minutes, filter about 5 ml of each solution through separate funnels into separate test tubes marked A and B. To each test tube add 1.0 ml of Ninhydrin reagent and gently swirl the tubes.

4. Make a water bath by adding 150 ml water to a 400 ml beaker and heat to boiling. Place test tubes A and B in this boiling water bath and heat until a color change (4 to 7 minutes) and remove from the heat. The Ninhydrin turns purple in the presence of amino acids but does not change color in the presence of protein.

5. For my results, tube A turned into a dark purple. Yet, tube B also turned into a lighter purple with some curd still at the sides of the tubes. I don't think tube B was supposed to turn purple right if no enzyme was added to it? If so, what may have caused this color change. ?

Answer 1

Nihydrin reacts with free amino acids only. In baker B (without enzyme), the boiling bath incubation might induce the breaking of some peptide bonds, allowing that some amino acid would be free in solution. That could be the reason of a light purple result instead of yellow.

If you incubate a protein more than 5 minutes in a boiling water batch , there is a small chance of breaking some peptide bonds besides the denaturation of the protein