LAB 4 : PROTEIN ANALYSIS

LAB 4 : PROTEIN ANALYSIS

Objectives:

u To determine protein concentration in various type of protein content

u To determine protein concentration using two types protein assays that are Biuret reagent.

Introduction :

Proteins are polymers of amino acids. Twenty different types of amino acids occur naturally in proteins. Proteins differ from each other according to the type, number and sequence of amino acids that make up the polypeptide backbone. As a result they have different molecular structures, nutritional attributes and physiochemical properties. Proteins are important constituents of foods for a number of different reasons. They are a major source of energy, as well as containing essential amino-acids, such as lysine, tryptophan, methionine, leucine, isoleucine and valine, which are essential to human health, but which the body cannot synthesize.

The biuret test (Piotrowski's test) is a chemical test used for detecting the presence of peptide bonds. In the presence of peptides, a copper(II) ion forms mauve-colored coordination complexes in an alkaline solution

Molecules with two or more peptide bonds react with Cu²⁺ ions in alkaline solution and form a purple complex. Nitrogen atoms of the peptide bonds form a coordination bond with the metal ion. The quantity of the complexes formed is proportional to the number of peptide bonds.

In practice, the determination of protein concentration is done using a calibration curve created using samples of known concentration. The protein treated with biuret reagent is measured at 540 nm after the purple product is formed.

The advantages of the method include that only few materials (e.g. Tris and amino acid buffers) interfere with it, it can be done in a short time and does not depend on the amino acid composition of the protein.

Image 1 : The characteristic color of a positive biuret test

Material and apparatus :

Stock solution of Bovine Serum Albumin (BSA) 10mg/ml

Deionized water (dH2O)

Test tubes and stand

Pipette

Biuret reagent

Spectrophotometer

Protein samp

Methodology :

(A) Preparation of biuret reagent

1. 300 mL of 10% (w/v) NaOH was added with stirred to 500 mL of a solution containing 0.3% copper sulphate pentahydrate  and 1.2% sodium potassium tartarate, then was diluted to 1 Liter.

(B) Protein preparation

1. 2 set of test tubes was prepared with the number 1 to 6 and the bovine serum albumin (BSA) stock solution (10 mg/ml) was prepared according to the concentration listed below:

Tube	BSA conc. (mg/ml)	H2O (ml)	BSA stock (ml)
1	0	1.0	0
2	1	0.9	0.1
3	2	0.8	0.2
4	3	0.7	0.3
5	4	0.6	0.4
6	5	0.5	0.5
7	6	0.4	0.6

2. The duplicate test tubes for protein samples were prepared and was pipetted 1ml of the protein samples carefully into each tubes carefully.

3. 2ml of biuret reagent was added to every tube : the 14 tubes for the standard curve and the duplicate tubes for protein samples.

4. The test tubes was covered with parafilm and was vortex briefly to ensure that the protein standards/ samples and the biuret reagent are thoroughly mixed.

5. The tubes was allowed to stand at 15 minutes.

6. The spectrometer was turned on and the wavelength was adjusted to 550nm.

(C) Determine protein concentration

1. 1ml of solution was transferred from tube 1 into a cuvette and was wiped gently with a paper towel to remove fingerprint and dust.

2. The absorbance was set to ‘zero’. This tube have been served as ‘blank’.

3. The absorbance of the other standards and sample protein were measured using the step as in (1). *DO NOT blank the instrument again.

4. The absorbance of each standards and samples were recorded.

5. A graph of standard curve was plotted using the absorbance value of protein standards and the absorbance values of the protein samples were interpolated.

 Result :

PROTEIN

PROTEIN I		PROTEIN II
SOY BEAN I	0.364	ALBUMIN I	0.579
SOY BEAN II	0.419	ALBUMIN II	0.513

SAMPLE OF PROTEIN

TUBE I		TUBE II
1	0.007	8	0.006
2	0.108	9	0.085
3	0.193	10	0.199
4	0.280	11	0.263
5	0.336	12	0.331
6	0.431	13	0.416
7	0.479	14	0.474

Discussion :

In this experiment, we used Biuret reagent test to test the content of protein in all those samples. The Biuret test is a chemical test used for detecting the presence of peptide bonds. In the presence of peptides, a copper(II) ion forms violet-colour coordination complexes in an alkaline solution. The Biuret reaction can be used to assess the concentration of proteins because peptide bonds occur with the same frequency per amino acid in the peptide. The intensity of the colour and hence the absorption at 540 nm is directly proportional to the protein concentration according to the Beer-Lambert law.

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.

Conclusion :

Throughout this lab, we can conclude that there are two ways of measuring protein concentration in a solution. It is either by using biuret assay or Lowry assay. But for thi experiment we only used Biurret reagent.  Both can be used to measure the protein concentration in a solution but the most accurate way to measure the concentration is by using Lowry assay. This is due to the fact that Lowry assay can measure the protein from as low as 1 μg/mL to 1500 μg/mL compared to biuret which can only measure the protein from 5 to 160 mg/mL. in this experiment, we had tested the protein concentration of albumin and soy bean.

References :

1) Nik Syarihah bt. Muhamad Zin, Siti Asilah bt. Abdul Rahman, Noorul Syafawani bt. Mohamed ,Rochelle Tang Nga Ning, Maisarah bt. Mokhtar, (2013, April 1 ) . Protein Experiment. Retrieved from http://biochemistrygirls.blogspot.com/2013/04/experiment-2-protein-experiment.html

2) Caprette David R. ( 2015, June 12 ). Biuret Protein Assay. Retrieved from https://www.ruf.rice.edu/~bioslabs/methods/protein/biuret.html

3) No Name. (No Date). Chapter 4. Spectrophotometry and protein concentration measurements. Retrieved from http://elte.prompt.hu/sites/default/files/tananyagok/IntroductionToPracticalBiochemistry/ch04s05.html