FACULTY OF SUSTAINABLE

AGRICULTURE

RT10303

CHEMISTRY FOR AGRICULTURE

DR. LUM MOK SAM

PRACTICAL TASK 5

(24/11/2020)

DETERMINATION OF PROTEIN IN PLANT SAMPLE

(LOWRY’S COLORICMETRIC METHOD)

Prepared by:

NOOR AMYRAH ALINA BT. MOHD AMERUL ‘ASRI BR20110034

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TITLE:

Determination of Protein in Plant Sample (Lowry’s Colorimetric Method)

OBJECTIVE:

To determine the protein in plant samples (Lowry’s Colorimetric Method)

INTRODUCTION:

Protein is a macronutrient that is essential for muscle mass building. It is commonly

found in animal products, although other sources, such as nuts and legumes, are also
present.

Three macronutrients are present: protein, fats and carbohydrates. Calories, or energy,
are given by macronutrients. To sustain life, the body needs large quantities of
macronutrients, hence the word "macro," according to the McKinley Health Center of the
University of Illinois. 4 calories are found in each gram of protein. Around 15 percent of the
body weight of a human is made up of protein.

The majority of individuals require 20 to 30 grams of protein per meal, Crandall said.
That's 2.5 egg whites at breakfast, for example, or 3 to 4 ounces of meat at dinner." She
said that at breakfast, most American women do not get close to adequate protein
anywhere." "That could impede their muscle mass, their metabolism, and their levels of
hormones."

APPARATUS:
1. Test tube
2. Distilled water
3. Volumetric flask
4. Filter
5. Pipette

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6. Distilling flask
7. Cuvette
8. Spectrophotometer

Reagents:

1. Phosphate Buffer : 0.1 M of pH 7.5

Firstly a 2.78 g of monobasic sodium phosphate in 100 ml distilled water was dissolved
and was categories as solution (a). Next, 5.365 g dibasic sodium phosphate Na2HPO4.7H20
or 7.17g of Na2HPO4.12 H20 in 100ml distilled water was dissolved and was categories as
solution (b). Then, in a volumetric flask, 16 ml of (a) solution and 84 ml of (b) solution were
mixed and diluted to 200ml with distilled water.

2. Alkaline Copper Sulphate Solution: (Fresh)

Firstly in a volumetric flask 3.0 g og Na3CO3 (Alkaline Sodium Carbonate) was dissolved
in 100 ml of 0.1 NaOH and was categories as solution (a). Next, 0.5 g of
CuSO45H20(Copper Sulphate Solution) in 100ml of 1% sodium potassium tartarate solution
was dissolved. Lastly, mixed 2 ml of solution (b) to 100 ml of solution (a) and shake well
before used them.

3. FolinCiocalteau Reagant : (Stock Solution)

There were also commercial solutions available that have been diluted with water (1:1)
before use. [Reagent in laboratory was prepared. 100 g sodium tungstate (NaWO4.2H2O) ,
700 ml water, 50 ml 85% phosphoric acid and 100 ml concentrated HCI in 1.5 litre
distillation flask were transferred. The mixture refluxed with condenser for 8-10 hours. 150
ml lithium sulphate, 50 ml water and few drops of bromine water were added. To remove
excess of bromine without condenser boiled them for 15 minutes. Diluted 1 litre and let it
cool. Lastly filtered (filtrate should not have greenish colour)]

4. Protein Standard Solution

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 50 mg of Bovine Serum Albumin (BSA) was dissolved in distilled water and was diluted
to a final volume of 50 ml in a volumetric flask. Further diluted 20 ml of this solution to 100
ml with distilled water in volumetric flask (200 mg/ml)

PROCEDURE:

Firstly, the standard protein solution were pipette out into six test tube that has
0,0.2,0.4,0.6, 0.8 and 1.0mL. Then, two test tube were set up for phenol which has a
reading 0.5mL for each test tube. After that, 1mL of distilled water were added to each test
tube. Then, 1mL of sample extract were added to each test tube and then 5mL alkaline
copper sulphate solution were added to each test tube and 0.5 ml of FolinCiocalteau
reagent (3) was mixed and added in all the tubes. Then, each test tube were mixed well 10
minutes. Leave the test tube for 20 minutes then set the reading of the spectrophotometer
to 750NM to read the absorbance of each test tube to get the results.. Lastly, the standard
curve with reading versus concentration of standard protein was drawn and the protein in
sample extract was determined with the corresponding readings.

RESULTS:

Reagents Test Tubes

1 2 3 4 5 6 7 8
Standard protein solution (mL) 0 0.2 0.4 0.6 0.8 1.0 0 0
Distilled water (mL) 1.0 0.8 0.6 0.4 0.2 0.0 0 0
Unknown sample (mL) 0 0 0 0 0 0 1.0 1.0
Alkaline copper sulphate solution (mL) 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0
FolinCiocalteau reagent (mL) 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5
Table 1: The experiment reagents set-up.

Reagents Tube 1 Tube 2 Tube 3 Tube 4 Tube 5 Tube 6

Standard protein solution (mL) 0.0 0.2 0.4 0.6 0.8 1.0
Standard protein solution (µg) 0 40 80 120 160 200

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Absorbance (750 nm) 0.0 0.077 0.156 0.202 0.268 0.297
Table 2: The reading of standard protein content (µg) and the absorbance 750nm.

Unknown sample Tube 7 Tube 8

Absorbance (750 nm) 0.190 0.184
Table 3: The absorbance (nm) of the unknown sample.

Tube 1 Tube 2 Tube 3 Tube 4 Tube 5 Tube 6

Standard protein content
0.0 40 80 120 160 200
(µg)
Absorbance (750 nm) 0.0 0.077 0.156 0.202 0.268 0.297
Table 4: The data needed for the standard curve

Calculation:
Standard Protein Content (µg)

200 µg/ml = 2 ml =200 µg

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Test Tube 1 = 0.0 x 200 = 0 µg

Test Tube 2 = 0.2 x 200 µg = 40 µg

Test Tube 3 = 0.4 x 200 µg = 80 µg

Test Tube 4 = 0.6 x 200 µg = 120 µg

Test Tube 5= 0.8 x 200 µg = 160 µg

Test Tube 6 = 1.0 x 200 µg = 200 µg

Absorbance of unknown sample

Test Tube 7 = 0.190

Test Tube 8 = 0.184

The average value of the unknown sample

(0.190+ 0.184)
=0.187
2

Dilution factor
Unknown sample Alkaline copper Folin-Ciocalteau Total volume
extract sulphate solution reagent
1mL 5mL 0.5mL 6.5mL

Total volume 6.5

Dilution factor = = ¿ 6.5
Initial volume 1.0
y=mx+C

C=0

0.187=0.05 x

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 0.187
x= =3.74
0.05

3.74 x 6.5=24.31 µg

DISCUSSION

In the experiment, the reagent of 5mL Alkaline copper, 0.5mL Folin-Ciocalteau and
Standard Protein Solution was used. This position was to engage the reactions of copper
ions and peptide bonds with the oxidation of aromatic protein residues in alkaline conditions.
The absorbance value was determined using a spectrophotometer set at 750nm. The value
of the straight line was determined from the graph by using regression in Excel. Y=0.05x is
the linear equation of the straight line given by the excel graph. A value of R2, which is
0.9472, has also been obtained. Finally, the Lowry process was based on the Cu+ reaction
formed by the oxidation of the peptide bonds with the reagent Folin-Ciocalteu. The reaction
produce the blue color.

CONCLUSION

In conclusion, the protein in the unknown sample is 24.31 µg. The standard curve
obtained is also acceptable to be R2 = 0.9472 with the line equation being y = 0.05x.

REFERENCES

Khanizadeh, S., Buszard, D and Zarkadas, C.G (1989). Seasonal variation of proteins and
amino acids

Sosulski, F.W. and Imafidon, G.I (1990). Amino acid composition and nitrogen-to-protein
conversion factors for animal and plant foods. J. Agric. Food.Chem., 38: 1351-1356.

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Sokal, R.R. and F.J. Rohlf. 1973. Introduction to Biostatistics. W.H. Freeman and Company,
San Francisco, California. 368 pp.