Kenyatta university biuret protein determination

Lab report on biuretic protein determination method

1. KENYATTA UNIVERSITY
SCHOOL OF MEDICINE.
DEPARTMENT OF MEDICAL BIOCHEMESTRY
NAME: LANDO ELVIS OTIENO
REG NO: P29S/16344/2015.
COURSE: MBCHB
LECTURER:DR OKUN/ Mr NJOGU STEPHENE
HANDING DATE: 5 /11/2015

SUBJECT:LABORATORY REPORT ON PROTEIN DETERMINATION
BY THE BIURET METHOD.


7/11/2015
P29S/16344/2015
Lando ELVIS O
©

2. BACKGROUND AND PRINCIPLE
-Proteins have a primary structure composed of amino acids linked by peptide bonds in a linear
manner.
The biuret reaction can be used for both qualitative and quantitative analysis of protein. The
biuret method depends on the presence of peptides bonds in proteins. When a solution of
proteins is treated with cupric ions (Cu2+) in a moderately alkaline medium, a purple colored
Cu2+ - peptide complex is formed which can be measured quantitatively by spectrophotometer
in the visible region. So, biuret reagent is alkaline copper sulfate solution.
Cu2+ - peptide complex

The intensity of the color produced is proportional to the number of peptide bonds that are
reacting, and therefore to the number of protein molecules present in the reaction system. The
reaction don't occur with amino acids because the absence of peptide bonds, and also that with
di-peptide because presence of only one peptide bond, but do with tri-, oligo-, and poly-
peptides. Biuret reaction needs presence of at least two peptide bonds in a molecule. The
reaction occurs with any compound containing at least two bonds of:
-HN-CO- , -HN-CH2- , -HN-CS-

3. In this experiment the amount of isolated protein is determined by biuret assay and from the
established standard curve . A standard curve is a quantitative research tool; it is a method of
plotting assay data that is used to determine the concentration of a substance like bovine
serum albumin protein. First you perform a biuret assay with various known concentrations of
protein(bovine serum albumin , 10mg/ml). The response is absorbance of the colored product.
Graph these data to make a standard curve, concentration on the X axis, and the absorbance on
the Y axis. Also perform the biuret assay with your sample. You want to know the concentration
of the protein in sample. To analyze the data, fit a line or curve through the standards. For the
sample, read across the graph from the spot on the Y-axis that corresponds to absorbance of
the sample until you intersect the standard curve. Read down the graph until you intersect the
X-axis. The concentration of protein in the sample is the value on the X-axis.
The biuret method has the advantage that it can be used in the presence of inorganic ions.
Normal human human protein concentration of normal plasma ranges from 50 – 100mg/ml.
Material and apparatus:
1. Protein sample of unknown concentration (bovine serum albumin , 10mg/ml)
2. Standard BSA (5 g/l)
3. Distilled water
4. Biuret reagent
5. Test tubes

4. 6. Label
7. Test tube rack
8. Pipettes
9. Pipette bulb
10. Vortex mixer
11. Spectrophotometer
12. Cuvettes
Methods:
1) Label 7 test tubes as (1 to 7) and place them in a test tube rack.
2) Add to each tube the solutions in the following table:
table 1 2 3 4 5 6 Serum
sample in
question(ml)
Standard
protein(10mg/ml)
0 0.2 0.4 0.6 0.8 1.0 1.0
Distilled
water(ml)
1.0 0.8 0.6 0.4 0.2 0 0
Biuret
reagent(ml)
3 3 3 3 3 3 3
Total mls 4 4 4 4 4 4 4
absorbance - - - - - - -

3) Mix well by vortex mixer and allow standing for 20 min.
4) Read the absorbance for each tube against the blank at 540 nm.
5) Calculate the protein concentration in each tube of standard.
6) Record your result in a table:
7) Plot the standard curve using concentration of standard tubes of BSA (mg/ml) against the
absorbance at 540 nm.
8) Calculate the mean of absorbance of the duplicate sample and obtain the concentration of
myoglobin in the sample from the standard curve.

5. 9) Find out the protein concentration (mg) in the original skeletal muscle tissue sample.

Result and observation
Test tube

Standard protein conc:mg/ml Absorbance at 540 nm

1 0 0.00
2 2 0.063
3 4 0.134
4 6 0.211
5 8 0.274
6 10 0.302
Serum sample in question(ml)
conc.
-? 0.124

Calculating the concentration of Serum sample in question(ml) conc.

Using the regression equation above:
Y=absorbance/optical density=0.124
X=? concentration of the protein
y = 0.0317x + 0.0054
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0 2 4 6 8 10 12
Absorbance
Protein Concentration (mg/ml)
Biuret method protein determination (by
lando elvis)

6. Therefore: 0.124= 0.031x + 0.005
X=0.119/0.031
X(concentration of the protein/serum) = 3.8388mg/ml
Dilution of the solution samples:

10times of dilution x 5times of dilution = 50 times dilution

The actual concentration of the protein in the samples:

3.8388mg/ml x 50times dilution = 191.935mg/ml.

DISCUSSION and CONCLUSION

The subunits which make up proteins are amino acids. The amino acids are joined together by
dehydration synthesis to forms chains, which are hundreds of amino acids long which is called
proteins. Proteins function as enzymes or as structural units in cells. They do most of the work
in a cell. Almost all of the exciting stuff such as metabolism, memory, hormone action, and
movement involves proteins. In this lab, we have learnt method of measuring protein
concentration, biuret assay.

The biuret reaction is a method that can be used to determine the amount of soluble protein in
a solution. The biuret reagent (copper sulfate in a strong base) reacts with peptide bonds
(which join amino acids to form proteins) and changes colour when it does so. The
spectrophotometer has been used to measure the intensity of the colour produced. The more
protein present the darker the colour.

In order to quantitatively determine how much protein is represented by a particular
absorbance reading it is necessary to construct a standard curve. This is done by performing the
biuret reaction on a series of prepared solutions of gelatin at 1,2,3,4,5 and 6 mg/ml in water.
The absorbance readings obtained from these solutions are used to construct a graph of
absorbance as a function of protein concentration. This graph is called the standard curve for
assay, and can be used to convert the absorbance readings for the experimental samples
(serum) into a protein amount or concentration.

Based on the graph that has been constructed, it shows that the standard protein
concentration for the sample as shown in the result. From the graph we can see that, there is
high concentration of serum protein in the sample compared to normal ranges. So, from the
experiment maybe there are some mistake happen while conducted it. That’s why our group
get the opposite result than the actual one. Error that occurred could be the mixing of using the
same pipette.

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eriment%20_.html
2. Anonymous,Biuret%20test%20-%20Wikipedia,%20the%20free%20encyclopedia.html
3. A Manual of Laboratory Techniques, 1983. Editors: Raghuramulu,n., Nair, M K and
Kalyanasundaram, S., National Institure of Nutrition, Hyderabad, India.
4 Plummer D.T., 1988. An introduction Practical Biochemistry,3
rd
edition, Tata Mc
Graw-Hill publishing company New Delhi, pp 159.
5. .Kenyatta university-laboratory-practical-manual- for-MBCHB-Year 1-semester1.