Lesson 1

Lesson 1: PROTEIN I.

Experiment 1: Ninhydrin reaction 1.Principle

tº Amino acid + ninhydrin  purple complex complex

Cu²+ Blue-purple complex (less stable )

Red(more

stable ) Amino acid used for test: monosodium glutamate

a. Procedures: i. Take a filter paper as required and make a small circle at each end of the paper ii. Add 1 drop of amino acid solution in each circle.dry the paper iii. Add one drop of ninhydrin solution in each circle.dry the paper iv.  Note down the color color immediately immediately v.  Now in one circle, add one drop of 5% CuSO4 solution vi. Dry the paper in oven vii.  Note down the color color immediately b. Result & explaination of experiment. experiment. 1. when we drop 1 drop of Nihydrin solution to the circle that had amino acids, and then dry the paper. The circles have the blue- purple color .(the color of Ruhemann’s

Purple- a chromophoric ninhydrin reaction) Reaction for explaination

compound-production

of

2.When we add 1 drop of 5% CuSO 4 solution to 1 circle and then dry the paper in oven: the color of the circle change from blue-purple into red- purple.  Because the Ruhemann’s purple is a unstable compound when the medium has the  presence 2 positive charged ions (Cu² +) it will be changed into the red-purple complex (stable form).

II.

Experiment 2: Xanthoprotein reaction a. Principle: This reaction tests for the presence of aromatic rings in proteins. This test is based on the ability of aromatic amino acids (tyrosine, tryptophan) to react with concentrated nitric acid to give dinitro derivates.

Note: Although Phenyl alanine is a aromatic amino acid, it do not have the Xanthoproteic reaction Amino acid (aromatic rings) + nitric acid

dinitro derivates  

quinoid

b. Procedure:

1.Take 5 drops of test solution in a test tube 2.Add 5 drops of concentrated nitric acid 3.Mix & observe the change of color 4. add 10% NaOH drop by drop & mixing thoroughly. The color intensities will change in the alakine medium 5.Note down the changes c. Result & explaination of experiment: i. When we add high concentrated nitric acid to test tube, HNO3 will react with the aromatic rings of the amino acids. Thus, the color of the solution change into yellow because: the aromatic ring of some amino acid residues in protein reacted to HNO3 to  produce the new production containing NO2 group that are  yellow. (the color of dinitro derivates the production of  Xanthoproteic reaction). ii. When we add NaOH to the test tube, NaOH will react with dinitro derivates and change it into quinoid form) so the solution will have the orange-yellow color (color of quinoid).

III. Experiment 3: Biuret reaction

a. Principle: The presence of peptide bonds can be test by their reaction with ions of copper salt in the alkaline medium, a Cu ²+-peptide complex which has blue-violet colour, is formed.

b. Procedures:

1.Take 5 drops of a protein solution in a test tube. 2.Add 2-5 drops of Biuret solution. 3.Mix & observe the change of colour. c. Result & explaination of experiment: Result: occur the blue-violet complex. Explain: When we add Biuret solution the 2 positive charged copper ion in alkaline medium (OH-) will react with peptide bonds of  proteins and will form the Biuret complex. The solution will have the blue-violet colour (the colour of Biuret complex).

IV. Quantitative determination of protein content by Biuret method a. Principle:

i. The Biuret complex can be quantitative measured by spectrophotomer in the visible region. It has the maximum absorbance at 540nm.

b. Procedures: i. Step 1: standard curve plotting No

Protein 0.1%(ml)

Distilled water (ml)

Biuret reagant (ml)

1

0.0

1.0

4

0.00

0.2625

2

0.2

0.8

4

0.02

0.3025

3

0.4

0.6

4

0.04

0.3694

4

0.6

0.4

4

0.06

0.5183

5

0.8

0.2

4

0.08

0.5672

6

1.0

0.0

4

0.10

0.6550

With:

X

OD

(%)

C is the protein concentration in each tube of standard

OD is the absorbance for each tube at 540 nm ii. step 2: dertermination of protein content in a given sample 1. Take a test tube and label it as 7 2. Add 1 ml of a protein solution with unknown protein content 3. Add 4 ml of Biuret reagant 4. Allow standing at room temperature for 30 min 5. Read the absorbance for the tube at 540 nm 6. Record the result 7. Determine protein content based on the standard curve c. Conclusion: the OD of tube 7 is 0,5525 . Thus, base on the standard curve we can said the protein content is about 0,0623%.