PROTIEN (AMENDED)

DEPARTMENT OF BIOLOGY

FACULTY OF SCIENCE & MATHEMATICS

UNIVERSITI PENDIDIKAN SULTAN IDRIS

SBK3013

PRINCIPLE IN BIOCHEMISTRY

EXPERIMENT 5: PROTIEN (AMENDED)

NAME	MATRIC NO.
MUHAMMAD FARIS BIN ISMAIL SAZEMI	D20141067089
MAYURIE PHUTHARANT A/P SURIN	D20141067078
NUR AFIQAH SYAHMINA BT MOHD KAMAL	D20141067091

GROUP: A

LECTURER’S NAME: DR. ROSMILAH MISNAN

INSTRUCTOR NAME: NUR ATIEKAH BT AZAHARI

INTRODUCTION

Determining the exact quantity of proteins in a solution is very often necessary in the biochemical practice. There are many ways to measure protein concentration. In chromogenic methods, the absorbance of a coloured product formed by the protein and an organic molecule is measured. Protein concentration can also be determined from the protein’s UV absorbance. However, these methods may give different results for different proteins of the same concentration. Different methods also can yield different results for the same protein. There is no absolute photometric protein concentration assay. All methods have advantages and disadvantages and we must choose among them by taking the following aspects into consideration: specificity, sensitivity, the measurable range of concentration, the accuracy, the nature of the protein to be examined, the presence of materials interfering with the measurement, and the time required for the measurement.

The first test is to determine the protein concentration by using the Biuret assay method. 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. Its disadvantages are its low sensitivity and that it requires at least 1 mg of protein.

Second test to determine the protein concentration is by using the Lowry assay method. The advantage of this method is Lowry assay is sensitive to low concentrations of protein. The major disadvantage of the Lowry method is the narrow pH range within which it is accurate. A variety of compounds will interfere with the Lowry procedure. These include some amino acid derivatives, certain buffers, drugs, lipids, sugars, salts, nucleic acids and sulphydryl reagents. The ammonium ions, zwitter ionic buffers, nonionic buffers and thiol compounds may also interfere with the Lowry reaction. These substances should be removed or diluted before running Lowry assays.

MATERIAL

·         Protein standard

·         Biuret reagent

·         Lowry reagent

·         Sample: fish, beef, chicken, peanut, green bean, soybean, red bean and dal bean

PROCEDURE

1.      Preparation of protein standard

1.      Solution of gelatin at 1, 2,3,4,5 and 6 mg/mL in water from the gelatin stock solution (10 mg/mL) for biuret assay was prepared.

2.      Solution of gelatin at 0.1, 0.2, 0.3, 0.4, 0.5 and 0.6 mg/mL in water from the gelatin stock solution (1 mg/mL) for Lowry method was prepared.

2.      Preparation of test samples

a.       Animal protein

1.      10 g of protein sample was weighted

2.      Macerated into smaller size

3.      Phosphate Buffer saline at 1:10 ratio was blended

4.      Sample was filtered by kitchen filter

5.      The supernatant was collected

6.      Sample was filtered again using Whatman filter No 1

7.      The supernatant was collected

b.      Plant protein

1.      10 g of protein sample was weighted

2.      The sample was crushed and grinded into fine paste or powder using mortar and pestle.

3.      The powder was dissolved in Phosphate Buffer saline at 1:10 ratio

4.      Sample was filtered by kitchen filter

5.      The supernatant was collected

6.      Sample was filtered again using Whatman filter No 1

7.      The supernatant was collected

3.      Protein assay

a.       Biuret assay

1.      0.5 mL of each protein was mixed with 2.50 mL of biuret reagent

2.      The absorbance of the samples at 540 nm after 10 minutes was measured

3.      The standard curved was plotted

4.      The protein content of the test sample was estimated using the standard curve.

b.      Lowry assay

1.      0.25 mL of each protein was mixed with 2.5 mL of  Lowry reagent 1

2.      The mixture was incubated at room temperature for 10 minutes.

3.      0.25 mL of Lowry reagent 2 was added and mixed immediately

4.      The mixture was incubated at room temperature for 30 minutes.

5.      The absorbance of the samples at 750 nm was measured.

6.      The standard curved was plotted

7.      The protein content of the test sample was estimated using the standard curve.

RESULT

Protein Standard

1.      Biuret

	Protein Number
B1	0.439
B2	0.509
B3	0.512
B4	0.542
B5	0.769
B6	0.995

2.      Lowry

	Protein Number
L1	0.132
L2	0.106
L3	0.406
L4	0.256
L5	0.115
L6	0.081

Protein Number for Other Sample

Sample	Biuret	Lowry
Beef	0.488	0.944
Fish	1.020	1.320
Braise Fish	1.138	0.406
Chicken	0.552	1.328
Soybean	0.447	0.377
Red bean	0.645	0.926
Peanut	0.476	1.219
Dal bean	0.746	0.214
Green bean	0.983	1.945

DISCUSSION

In Biuret assay we combine protein samples with Biuret Reagent which contains copper ions in a basic solution. The copper ions will complex with the amide groups in the proteins to create a blue colour that will be measured using a spectrophotometer. The amount of blue colour that forms is directly proportional to the quantity of protein in the sample. In order to determine the actual concentration of protein in the unknown sample it is necessary to graph the standard curve. Our standard graph is shown as above.

The Lowry assay 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 used with protein concentrations of 0.01–1.0 mg/Ml. It is based on the reaction of Cu+, produced by the oxidation of peptide bonds, with Folin–Ciocalteu reagent. The concentration of the reduced Folin reagent is measured by absorbance at 750 nm. As a result, the total concentration of protein in the sample can be deduced from the concentration of Trp and Tyr residues that reduce the Folin–Ciocalteu reagent.

In our experiment, the results for Biuret test showed that braise fish has the highest protein number of 1.138 followed by fish, green bean, dal bean, red bean, chicken, beef, peanut and soybean. In the other hand, Lowry test showed that green bean has the highest protein number of 1.945 followed by chicken, fish, peanut, beef, red bean, braise fish, soybean and dal bean.

There are few errors occur in our experiment. First and foremost, the measuring cylinder that have been used to measure volume each samples are not cleansed thoroughly, and this may due to some particles left can alter the measurement of each sample. Furthermore, the absorbance of samples are measured less than 10 minutes. This may cause the reading of the absorbance are not accurate.

QUESTION AND ANSWER

1.      3 alternative methods of determining protein concentration are

i)                    By using UV absorbance. Protein concentrations can be determined directly by ultraviolet spectroscopy because of the presence of tyrosine and tryptophan which absorb at 280 nm. Furthermore, this method include high sensitivity so valuable protein samples can be recovered.

ii)                  BCA Protein Assay. Bicinchoninic acid (BCA) reacts with cuprous ions to generate purple colour at 562 nm. Cuprous ions are produced by the reduction of cupric ions by proteins in alkaline solutions. It is compatible with a large number of extraneous materials found in protein preparations.

iii)                Dye-binding method. Dye Coomassie Blue G-250 is dissolved in an acidic solution causing it to absorb at 465 nm (reddish brown). When the dye (negatively charged) binds to the positively charged protein molecule the absorbance undergoes a shift to 595 nm (blue). This shift in absorption maximum is proportional to protein concentration over a broad range.

2.      Appropriate blank is a reference measurement of the transmitted light as a function of wavelengths is stored in memory. When a measurement of a sample is made, the intensity of light that has been transmitted through the sample is recorded. Appropriate blank is needed to calibrate the spectrophotometer. This is because it may be some other particles or substances presence in the sample.

CONCLUSION

In conclusion, for the Biuret test, braise fish has the highest protein number that is 1.138 meanwhile for the Lowry test, green beans shows the highest protein number that is 1.945 when compared to the other sources of protein.

REFERENCES

N. A. Khan and K. N. Singh. 2014. Laboratory manual of biochemistry. New Delhi: Daya
             Pub. House

Moran, Laurence A., Horton, H. Robert, Scrimegeour, K. G. and Perry, Marc. 2014. Principle
               in Biochemistry. 5^th ed. Upper Saddle River, NJ: Prentice Hall.