UNIVERSITI PENDIDIKAN SULTAN IDRIS35900 TANJONG MALIM, PERAK DARUL RIDZUAN

FAKULTI :SAINS DAN TEKNOLOGI

PRINCIPLE IN BIOCHEMISTRY (SBK3013)

EXPERIMENT 2:PROTEIN ANALYSIS

PREPARED BY :UMI ABIBAH BT SULAIMAN D20091034811SITI RAHAYU BT MOHAMED NOOR D20091034855AZMA AMIRA MOHAMAD D20091034859NUR AFIQAH BT MUHAMAD APANDI D20091034872AMEERA BT YAHYA D20091034814

SEMESTER 8SESI 2009/2010

LAB SESSIONTHURSDAY(10.30 A.M. - 1.30 P.M.)

LAB INSTRUCTORDR ROSMILA MISNAN

PROTEIN EXPERIMENT

Experiment 2: The determination of three protein samples using Biuret and Lowry Assay.

Objective:

1. To learn the principles of protein assays.

2. To determine protein concentrations of protein samples using the Biuret Protein Assay

and Lowry assay.

Introduction:

The determination of protein concentration is an essential technique in all aspects of

protein studies.This lab activity is designed to teach students the principles behind a common

protein estimation assay known as the BiuretProtein Assay and Lowry Protein

Assay.Although there are a wide variety of protein assays available none of the assays can be

used without first consideringtheir suitability for the application. Each method has its own

advantages and limitations and often it is necessary to obtainmore than one type of protein

assay for research applications.

In the copper ion based protein assays, protein solutions are mixed with an alkaline

solution of copper salt, cupricions (Cu2+). The protein assay is based on the interaction of

cupric ions with protein in an alkaline solution and is commonlyreferred to as the Biuret

assay. The interaction of cupric ions (Cu2+) with protein results in a purple color that can be

read at 540nm. The amount of color produced is proportional to protein concentration.

The Lowry protein assay method for protein concentration determination is one of the

most venerable and widely-used protein assays. Hydrolysis is probably the most accurate

method of determining protein concentration  followed by amino acid analysis. Most other

methods are sensitive to the amino acid composition of the protein, and absolute

concentrations cannot be obtained. The Lowry procedure is sensitive, and is moderately

constant from protein to protein. The Lowry protein estimation  has been so widely used that

it is a completely acceptable alternative to a rigorous absolute determination in almost all

circumstances in which protein mixtures or crude extracts are involved.

Result:

Biuret Assay

Table 1.1: Biuret assay

Concentration (mg/ml) Absorbance (nm)

Blank 0.00

1 0.138

2 0.139

3 0.190

4 0.192

5 0.194

6 0.211

Table 1.2: Determination using Biuret assay

Samples Absorbance before

dilution (nm)

Dilution Absorbance after dilution

(nm)

Concentration(mg/ml)

Chicken egg 0.936 1mL of sample + 9 mL of distilled water

0.106 2.0 x 10=20

Quail egg 0.870 1mL of sample + 9 mL of distilled water

0.090 1.6 x 10= 16

Duck egg 0.764 1mL of sample + 9 mL of distilled water

0.066 1.0 x 10= 10

Chicken egg(ayam kampung)

0.2301mL of sample + 9 mL of distilled water

0.195 1.8 x 10= 18

Omega egg 0.256 1mL of sample + 9 mL of distilled water

0.197 1.9 x 10= 19

Graph of absorbance value (nm) versus gelatin concentration (mg/ml) for Biuret assay

Lowry Assay

0 1 2 3 4 5 6 70

0.05

0.1

0.15

0.2

0.25

Y-Values

Gelatin concentration (mg/ml)

abso

rban

ce v

alue

(nm

)

Table 2.1: Lowry assay

Concentration Absorbance (nm)

Blank 0.00

0.1mg/mL 0.053

0.2 mg/mL 0.133

0.3mg/mL 0.143

0.4 mg/mL 0.2070.5mg/mL 0.185

0.6 mg/mL 0.166

Table 2.2: Determination using Lowry assay

Samples Dilution Absorbance after dilution

(nm)

Concentration(mg/ml)

Chicken egg 1mL of sample + 9 mL of distilled water

0.072 0.16 x 10=1.6

Quail egg 1mL of sample + 9 mL of distilled water

0.134 0.3x 10= 3.0

Duck egg 1mL of sample + 9 mL of distilled water

0.185 0.56 x 10= 5.6

Chicken egg(ayam

kampung)

1mL of sample + 9 mL of distilled water

0.173 0.41 x 10= 4.1

Omega egg 1mL of sample + 9 mL of distilled water

0.131 0.25 x 10= 2.5

Graph of absorbance value (nm) versus gelatin concentration (mg/ml) for Lowry assay

RESULT SUMMARY

0 0.1 0.2 0.3 0.4 0.5 0.6 0.70

0.05

0.1

0.15

0.2

0.25

Y-Values

Gelatin concerntration (mg/ml)

Abso

rban

ce v

alue

(nm

)

Table 3.1 Protein concentration obtained from Biuret assay and Lowry assay

Type of sample Concentration (mg/ml)

Biuret Assay Lowry AssayChicken egg 20 1.6

Quail egg 16 3.0Duck egg 10 5.6

Chicken egg(ayam kampung)

18 4.1

Omega egg 19 2.5

Discussion:

In this experiment, we had used Biuret assay and Lowry assay to determine protein

concentration in five protein food samples which are chicken egg, quail egg, duck egg,

chicken egg (ayam kampung) and omega egg.

Biuret test indicate the presence of peptide bond between the amino group and the

carboxyl acid group on adjacent amino acids in a protein. The violet colour is a positive test

for the presence of protein. The greater the violet colour intense, the greater the number of

peptide bonds that reacts.

Biuret test also has its limitations and not very sensitive which are the Biuret only test

whether peptide bonds in protein are present in a sample, it will not determine how much

there is unless we compare our sample of unknown concentration with a standard of known

protein concentration.

However biuret test will not detect free amino acids and the characteristic purple

colour will not show up if we only have small peptides, since the number of peptide bonds

may not be sufficient to yield colour. The Biuret assay is not much good for protein

concentrations below about 5 mg/ml. By using the Folin‐Ciocalteu reagent to detect reduced

copper makes the Lowry assay nearly 100 times more sensitive than the Biuret reaction alone.

Based on the result obtained we had plotted the standard curve by using Biuret assays,

and we compare with the five type of albumin sample. We found that the absorbance value of

these five types of albumin sample is higher than the standard curve. Then the five must do

the dilution to lower the reading from the standard curve. For dilution, the ratio used is 1 ml

of protein samples to 9.0 ml of distilled water. After dilution, we get the results which are the

protein concentration in chicken egg is the highest which is 20 mg/mL, the second higher of

protein concentration by using Biuret assays is omega egg which is 19 mg/mL and the third

higher protein concentration using Biuret assays is chicken egg(ayam kampung) which is

18mg/mL. Quail egg show the protein concentration 16mg/mL and the lowest protein

concentration by using Biuret assays is duck egg which is 10 mg/mL.

Next we had carried out Lowry assay to determine the protein concentration in three

protein samples. The Lowry method is more sensitive since it combines the reactions of

copper ions with the peptide bonds under alkaline conditions with the oxidation of

aromatic protein residues. The Lowry method is best used with protein concentrations of 0.01-

1.0 mg/mL and is based on the reaction of Cu+, produced by the oxidation of peptide bonds,

with Folin-Ciocalteu reagent.

The following substances are known to interfere with the Lowry assay are detergents,

carbohydrates, glycerol, potassium compounds, sulfhydryl compounds, most phenols, uric

acid, guanine, and calcium. Many of these interfering substances are commonly used in

buffers for preparing proteins or in cell extracts.

From the result of Lawry assays we also had been plotted standard curve graph, and

we compare with the five type of albumin sample. For dilution, the ratio used is 1 ml of

protein samples to 9.0 ml of distilled water. After dilution, we get the results which are the

protein concentration in duck egg is the highest among the other four which is 5.6 mg/mL, the

second higher of protein concentration by using Lowry assays is chicken egg (ayam

kampung) egg which is 4.1 mg/mL and the third highest protein concentration using Lowry

assays is quail egg which is 3.0 mg/mL. Omega egg shows the protein concentration

2.5mg/mL and the lowest protein concentration by using Lowry assays is chicken egg which

is 1.6 mg/mL.

When compare the result of Lowry assay with biuret essay, there is some differences.

This is might be because of some errors that occur while we conduct our experiment. For

instance we just estimate the volume of the sample when we poured into the vial. It should an

actual volume that is same for all protein. Second, there might be error when we dilute the

protein, caused by parallax error when reading the measuring cylinder.

As the conclusion, Lowry assays is best method in determining the protein

concentration because it is best used with protein concentrations of 0.01-1.0 mg/mL which is

suitable when test with 0.25 mL of protein sample of albumin of chicken, duck, quail, ayam

kampong and omega which is in the range of 0.01-1.0 mg/mL. Meanwhile, Biuret assay is not

very efficient in determining the protein concentration as it is not much good for protein

concentrations below about 5 mg/ml. Thus, albumin of duck has the highest protein

concentration, albumin of ayam kampong is the second highest in protein concentration,

albumin of quail has the third highest in protein concentration, omega chicken has the fourth

highest in protein concentration and the last one is albumin of chicken.

Instead of these two method, there are several method can be used in determining

protein concentration. First is The Bradford assay, a colorimetric protein assay, is based on

an absorbance shift of the dye Coomassie Brilliant Blue G-250 in which under acidic conditions the

red form of the dye is converted into its bluer form to bind to the protein being assayed. It also has

disadvantages which is the Bradford assay is linear over a short range, typically from 0 µg/ml to

2000 µg/ml, often making dilutions of a sample necessary before analysis. It is also inhibited by the

presence of detergents.

Conclusion

1. Protein concentration can be determined using Biuret protein assay and Lowry protein

assay.

2. The protein concentration in chicken egg is 20mg/mL , omega egg is 19 mg/mL,

chicken egg(ayam kampong) is 18mg/mL, quail egg is 16mg/mL and duck egg is

10mg/mL based on Biuret method.

3. The protein concentration in duck egg is 5.6mg/mL , chicken egg(ayam kampong) is

4.1 mg/mL, quail egg is 3.0mg/mL, omega egg is 2.5mg/mL and chicken egg is

1.6mg/mL based on Lowry method.

4. Protein concentration can be found out from the standard curve.

References

1. www.gbiosciences.com/.../633453707995878750.pdf

2. wolfson.huji.ac.il/purification/PDF/Protein.../PIERCE_BIURET.pdf

3. biochemistry.musc.edu/.../Lowry%20Protein%20Assay... - United States

4. www.molecularstation.com/protein/lowry-protein-assay/