~ Experiment 2: Protein Experiment ~

TITLE

Experiment 2: Protein Experiment

OBJECTIVES

1.    To determine protein concentration in various type of protein content

2.    To determine protein concentration using two types protein assays that are Biuret assay and Lowry assay.

INTRODUCTION

In the experiment to determine the protein concentration, Biuret assay and Lowry assay is used.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-coloredcoordination complexes in an alkaline solution. Several variants on the test have been developed, such as the BCA test and the Modified Lowry test. 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 color, and hence the absorption at 540 nm, is directly proportional to the protein concentration, according to the Beer-Lambert law. Despite its name, the reagent does not in fact contain biuret ((H₂N-CO-)₂NH). The test is so named because it also gives a positive reaction to the peptide-like bonds in the biuret molecule.

While, the Lowry protein assay is a biochemical assay for determining the total level of protein in a solution. The total protein concentration is exhibited by a color change of the sample solution in proportion to protein concentration, which can then be measured using  colorimetric techniques. It is named for the biochemist Oliver H. Lowry who developed the reagent in the 1940s. His 1951 paper describing the technique is the most-highly cited paper ever in the scientific literature, cited over 200,000 times.  The method combines the reactions of copper ions with the peptide bonds under alkaline conditions (the Biuret test) with the oxidation of aromatic protein residues. The Lowry method is best used with protein concentrations of 0.01–1.0 mg/mL. Lowry test is measured for the absorbance at 750 nm.

PROCEDURE

Dilution

1.    Protein is diluted for 10% and sample is diluted for 100% in order to make sure the validity of protein absorbance for protein test.

2.    Protein with 5 different volume that are 1mg/mL, 2 mg/mL, 3mg/mL, 4mg/mL, 5mg/mL and 6mg/mL is diluted for 10% by mixing 1mL of protein with 9mL of distilled water.

3.    Protein sample with 5 different protein sample that are duck egg, chicken egg, omega egg, puyuh egg, and ayam kampung egg firstly is diluted for 10% by mixing 1ml of protein sample with 9mL of distilled water.

4.    The diluted protein sample is then diluted for another 10% by mixing 1 ml diluted protein sample from the first dilution with 9ml of distilled water.

a    Biuret assay

                                                         

1.    The experiment is done with 5 different 10% diluted protein that are 1mg/mL, 2 mg/mL, 3mg/mL, 4mg/mL, 5mg/mL and 6mg/mL .

2.    The experiment also repeated by replacing protein with 5 different protein sample that have been diluted for 100 % that are duck egg, chicken egg, omega egg, puyuh egg, and ayam kampung egg.  

b      Lowry assay

RESULT

1. Standard Protein

Standard protein (mg/ml)	Absorbance (nm)
1	0.155
2	0.193
3	0.210
4	0.273
5	0.317
6	0.357

2. Samples Protein

Types of sample egg	Absorbance based on graph (nm)	Protein sample (mg/ml)
Duck	0.273	4.00
Chicken 1 (Normal Chicken)	0.331	5.35
Chicken 2 (Ayam Kampung)	0.298	4.50
Omega 3	0.214	2.60
Quail (Puyuh)	0.253	3.50

Graph and Calculation

                                                                      Full view

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A) Biuret assays

Dilution of the solution samples:

The actual concentration of the protein in the samples:

a)    Omega 3

= 2.6 mg/ml × 50 times of dilution

= 175 mg/ml

b)    Quail

= 3.5 mg/ml × 50 times of dilution

= 175 mg/ml

c)    Duck

= 4.0 mg/ml × 50 times of dilution

= 200 mg/ml

d)    Chicken 1 (ayam biasa)

= 4.5 mg/ml × 50 times of dilution

= 225 mg/ml

e)    Chicken 2 (ayam kampung)

= 5.35 mg/ml × 50 times of dilution

= 267.5 mg/ml

DISCUSSION

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 (eggs from duck, chicken 1 (ayam biasa), chicken 2 (ayam kampung), omega 3, quail) into a protein amount or concentration.

Based on the graph that has been constructed, it shows  that the standard protein  concentration for the samples as shown in the result. From the graph we can see that, the highest concentration of protein is chicken 1 and the lowest is omega 3. But for the real result actually the omega 3 will get the highest and the chicken1 will get the lowest. 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. 

Lowry method is one of the ways to determine the protein concentration in a solution. However it needs two-step procedure which requires a minimum of 40 minutes incubation time. The sensitivity of Lowry Assay reagent is greatly enhanced over that of the Biuret total protein reagent. The working range of the method covers the total protein rage from 1 to 1500 μg/mL. However, the working range for Biuret assay is from 5 to 160 mg/mL. The reading of colour at 750 nm for Lowry Assay is recommended because at this wavelength, few other substances absorb light. The amount of light absorbed at 750 nm is directly proportional to the amount of protein in the sample.

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.  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 in five different types of eggs, which are, duck’s egg, ayam biasa ( chicken 1 ) and ayam kampung ( chicken 2 ), omega 3 egg and quail’s egg. From the graph plotted, chicken 1 contain the most protein while omega 3 contain the least. The results supposed to be vice versa, omega 3 should contain the most protein whereas chicken 1 the least. This happened due to some mistakes we make during pipetting the samples.

REFERENCES