Google Search

Friday, April 1, 2011

ABSORPTION SPECTRA OF PROTEINS IN ULTRA-VIOLET RANGE


    ABSORPTION SPECTRA OF PROTEINS IN ULTRA-VIOLET RANGE
Aim:
To obtain absorption spectra of protein in the Ultra-Violet range.
Principle: 
Ultra violet spectroscopy is concerned with the study of absorption of UV radiation which ranges from 200 to 400 nm. Compounds which are colored absorb radiation from 400 to 800 nm. But compounds which are colorless absorb radiation in the UV region. In both UV as well as visible spectroscopy, only the valence electrons absorb the energy, thereby the molecule undergoes transition from ground state to excited state. This absorption is characteristic and depends on the nature of electrons present. The intensity of absorption depends on the concentration and path length as given by Beer-Lambert’s law.
The types of electrons present in any molecule may be conveniently classified as
1.      ‘σ ' electrons: these are the ones present in saturated compounds. Such electrons do not absorb near UV, but absorb vacuum UV radiation (<200nm).
2.      ‘π ‘electrons : These electrons are present in unsaturated compounds (eg) double or triple bonds.
3.      ‘n' electrons: these are non bonded electrons which are not involved in any bonding (eg) lone pair of electrons like in S, O, N and Halogens.
Below 230 nm, the extinction of a protein solution rises steeply reaching a maximum at 190 nm, this is mainly due to the peptide bond. In practice, it is more convenient to measure the extinction at 210 nm where the specific extinction coefficient is about 200 for most proteins. All proteins here since the peptide bond content is similar.
Tyrosine and tryptophan absorb at 275 nm and 280 nm and so proteins containing these amino acids will also absorb in this region. The specific extinction coefficient varies according to how much of these amino acids are present in the particular protein.
Chemicals required:
1.      Standard Bovine serum albumin
2.      9% saline solution
3.      Protein in serum diluted 1: 10,000 with 9% saline solution
4.      Dis.H2O
Apparatus required:
1.      Spectrophotometer
2.      Glass Cuvettes 
3.      Standard flasks, etc
Procedure:
Dissolve 10mg of Bovine Serum Albumin in 100 ml.distilled water. Add a few drops of 0.1N NaOH to preserve the solution. Prepare the following concentrations of Bovine Serum Albumin: 2mg%, 4mg%, 6mg%, 8mg%, and 10mg% as given below:
Test Tube No.
10mg% of Bovine Serum Albumin in ml.
Distilled Water in ml
Concentration in mg%
1 (Blank)
-
10
-
2
2
8
2
3
4
6
4
4
6
4
6
5
8
2
8
6
10
0
10
7( Unknown)
5
5
?
 
Now measure the extinction of all the solutions at 210 nm taking distilled water as blank and adjusting the spectrophotometer to 100% T with blank. Plot a graph of extinction against protein concentration (in mg %). You get a straight line graph passing through the origin. Determine the concentration of protein in the unknown solution by extrapolation.
 Observations:
Test Tube No.
Protein Concentration in mg%
Extinction
1
-

2
2

3
4

4
6

5
8

6
10

7
-


 Calculations:
1.      From Observations:
Extinction ‘A’ = 6 mg % of proteins
Extinction ’B’ =  mg% of protein
1ml .of unknown solution =  mg% of protein
Protein concentration in the unknown solution is= …….mg%
2.      From Graph:
The concentration of protein in the unknown solution =…….mg%
Results:
1). From Calculations:
  Amount of protein in the unknown sample=…….. gm%
2). From Graph:
Amount of protein in the unknown sample = ……..gm%


0 comments:

Post a Comment