EXPERIMENT # 2

Extraction and characterization of albumin form egg and casein from

milk Agudia, Myrna B. │Begino, Vianney Frances H. │Guerrero, Lara Marie C. │Lacdao,

May Q. │ Valmores, Azriel T.

Abstract Protein isolation and characterization techniques serve its relevance in the study of protein with

respect to its function, structure, and interactions with other molecules. This experiment was focused on

two proteins found in food, albumin and casein. Albumin and casein were extracted from egg and milk,

respectively, and their concentrations were determined with the use of the Warburg-Christian method and

Bradford Assay. Two trials for each method were performed. From the Warburg-Christian method, the

resulting average concentrations of albumin and casein were 0.4501 and 0.3323, respectively. Isolation

and characterization of proteins would have to be done using a more effective method to ascertain accuracy

of results.

INTRODUCTION

Protein isolation is a method of

separating a single type of protein from

its natural source or from a mixture of

several types of proteins. This process is

important in studying the function of a

specific protein, its structure and its

interaction with other materials in the

human body.

Extraction of protein from its

source requires breaking the tissue or

cell containing it and immersing it into a

solution. In this process, the tissue or

cell undergoes different procedures like

freezing, sonication, homogenization,

filtration, and permeabilization by an

organic solvent and after the soluble

protein has been separated from the

insoluble type, the protein of interest can

be isolated from the cell membrane or

DNA by centrifugation.

Protein can be further purified

by using different techniques such as

chromatography, centrifugation,

filtration and electrophoresis. When

isolating and characterizing proteins,

they must be stabilized to avoid

denaturation. Proteins can get denatured

at a high temperature as such it must be

kept at a fairly cool temperature.

The pH should be maintained to

inhibit proteases which destroy small

peptide bonds and proteins must be kept

at a high concentration, because many

proteins are unstable at air-water

interfaces or at low concentration.

Protein concentration may be

estimated using two spectrophotometric

methods-the Warburg-Christian method

and the Bradford Assay. In the first

method, protein concentration is

estimated by making a direct absorption

measurement of the solution in the UV

range. Protein has its maximum

absorbance at 280 nm due to the

absorption of their intrinsic tyrosine and

tryptophan amino acid residues. This

method is good because it is direct and

nondestructive. However the amount of

tyrosine and tryptophan vary in each

protein so this is only applicable for

semi-quantitative analysis of protein

sample. The Bradford Assay or protein-

dye assay uses the shift in the maximum

absorption of the Coomassie Brilliant

blue dye from 465nm to 565nm under

dilute acid solution to indirectly estimate

the amount of protein in the sample. It is

also advantageous, because it is easy to

perform and there are only few

interfering substances. Furthermore, it is

the high concentration of detergents that

disrupt the binding of the dye to protein

because of their amphipatic nature.

This experiment endeavors to

isolate casein from milk and albumin

from egg using some methods employed

in protein isolation and then to apply the

spectrophotometric methods in

characterizing and estimating the

concentrations of the extracted proteins.

METHODOLOGY

A. Extraction of Proteins

For the extraction of albumin

from an egg, 20 ml of egg white was

measured and then placed in a beaker.

After stirring the egg white with a

stirring rod, 2ml of 1.0 M HOAc was

added, dropwise, then the resulting

mixture was filtered using a cheese cloth

and an equal volume of saturated

ammonium sulfate solution was added to

the filtrate. The mixture was left to stand

for 30 minutes then it was placed in the

centrifuge and the precipitate was

discarded. The supernatant was then

transferred into a 250-ml Erlenmeyer

flask immersed in an ice bath. Next,

50% saturated ammonium sulfate was

added until turbidity persisted. The

mixture was allowed to stand for 15

minutes for complete precipitation and

was centrifuged again. The resulting

supernatant was discarded and the

precipitate was collected and weighed.

Then 20 ml of 10% (w/v) solution of

albumin in 0.9% NaCl was prepared

from this and stored in the refrigerator

for future experiments.

For the extraction of casein from

milk, 25 ml of of fresh milk was

measured and 0.1 M HCl was added

dropwise to it until a flocculent

precipitate was formed and the pH was

decreased to 4.8. The resulting mixture

was placed in the centrifuge and the

supernatant was discarded afterwards.

Then 95% ethanol was added to wash

the residue. It was centrifuged again and

the ethanol washings were decanted. The

resulting precipitate was washed with

acetone and was air dried under the

hood. The crude precipitate was weighed

and out of this, 20 ml 1% (w/v) casein

solution was prepared and then stored in

the refrigerator for the next experiments.

B. Determination of Protein

Concentration

Due to the unavailability of the needed

reagents, the determination of protein

concentration in the laboratory was not

performed instead a dry laboratory was

done using the data given by the

instructor.

RESULTS AND DISCUSSION

This experiment is composed of

two parts. First part is extraction of

proteins and the second is determining

protein concentration.

The experiment started with

extraction of proteins needed for the

experiment. Two common proteins were

extracted, namely egg albumin and milk

casein. In extraction of egg albumin and

casein, saltation using ammonium

sulfate and acid respectively was

conducted. The egg albumin yielded in

our extraction weighed less than 0.1

grams while casein weighed 2.1 grams.

In extraction of egg albumin,

saturated ammonium sulfate, estimated

25% (w/v), and 50% saturated

ammonium sulfate, estimated 13% (w/v)

was used. The egg white solution with

acetic acid was added first with saturated

ammonium sulfate then centrifuged then

added with 50% ammonium sulfate

again and centrifuged. The principle of

adding ammonium sulfate and

centrifuging it is the formation of

precipitate of proteins that precipitates at

certain percent and below and separating

it to other proteins that did not

precipitate at such concentration via

centrifugation.

Salting-out process starts with

the determination of the percentage of

ammonium sulfate of which the desired

protein precipitates the most. The protein

solution with impurities is first added

with the concentration of ammonium

sulfate lower than the optimal

concentration needed for the

precipitation of the desired protein. After

that, the solution will be centrifuged to

separate the precipitate and the

supernatant. Since the desired protein

will not yet be precipitated, the first

precipitate will disposed and the

supernatant will be added with a higher

concentration of ammonium sulfate, of

which it will achieve the concentration

that is optimal for precipitating the

desired protein, and will be centrifuged

again for the separation of the filtrate

and precipitate. This time, the filtrate

will be disposed while the precipitate

will be stored because the desired

protein was already in the precipitate.

According to Chick and Martin,

in their journal article named “The

Precipitation of Egg-Albumin by

Ammonium Sulphate. A Contribution to

the Theory of the “Salting-out” of

Proteins”, egg albumin concentration in

the filtrate is highest when concentration

of ammonium sulfate is 22% - 23%

(w/v). Any increase in ammonium

concentration will yield to the

precipitation of the desired protein. In

this experiment, the egg white solution

was subjected to saturated ammonium

sulfate of which the concentration is

25% (w/v). This implies that after

adding the ammonium solution, the

desired protein, of which is egg albumin,

is precipitated in the solution.

Unfortunately, the first precipitate after

the centrifugation is disposed. This may

have caused the low yield in terms of

weight of egg albumin in the extraction

of egg albumin in egg white. Added to

that, the egg albumin concentration in

the egg white may have caused the low

yield in weight value/

In the extraction of casein from

milk, 0.1M HCl and 95% ethyl alcohol

was used. The fresh milk was added with

0.1M HCl dropwise until pH is

approximately 4.8 and then centrifuge.

The filtrate obtained was disposed and

the precipitate is collected and was

added with 95% ethyl alcohol. The

solution was then centrifuged and the

precipitate was collected and was further

subjected to purification.

The addition of 0.1M HCl, or an

acid, is like the saltation process using

ammonium sulfate. Casein is insoluble

in certain acidic conditions but tends to

dissolve in much acidic conditions that is

why the pH of the milk solution is

monitored in extracting casein. After

that, the solution is centrifuged to

separate the precipitate and the filtrate.

Since casein has already precipitated, the

filtrate was disposed and the precipitate

was collected. The collected precipitate

at this point is not yet purely casein so

95% ethyl alcohol was added to the

precipitate, of which casein is insoluble,

to dissolve other proteins that have

precipitated also upon the addition of

0.1M HCl. The solution was then again

centrifuged to separate the filtrate and

the precipitate. The filtrate was then now

disposed and the precipitate was washed

with acetone and air-dried.

Saltation is one way only to

extract and purify proteins. Other ways

of protein extraction and purification are

through dialysis, ultrafiltration and

desalting columns. The use of dialysis

separates smaller from larger molecules

because of the minute-sized pores found

in the semi-permeable membrane of the

dialysis bag. The smaller molecules are

made to flow freely through the

membrane. Ultrafiltration also separates

smaller from larger molecules except

that the small molecules are forced to

pass through the membrane. Lastly, the

use of desalting column separates not

only the small molecules from big ones

but also with minute molecules with low

molecular weight.

The second part was the

determination of protein concentration.

The protein concentration will be

determined using two common assays on

finding protein concentration: the

Warburg-Christian Method and Bradford

Assay, both using spectrophotometer.

The Warburg-Christian Method uses the

equation:

Protein Concentration (mg/mL) = 1.55A280 – 0.76A260

while Bradford Assay uses the plot of

absorbance versus concentration of a

standard to predict and determine protein

concentration of sample.

Unfortunately, this part was not

conducted because of technical

problems. However, made up data was

given for analysis. The data is as

follows.

Albumin Casein

Trial 1 Trial 2 Trial 1 Trial 2

A280 0.5786 0.4935 0.4171 0.4169

A260 0.5204 0.4817 0.4127 0.4138

A280/A260 1.1118 1.0245 1.0107 1.0075

Average

Ratio 1.0712 1.0712 1.0091 1.0091

Protein

concentration

(mg/mL)

0.5013 0.3988 0.3329 0.3317

Table1. Warburg-Christian Method. Bold

values are derived from data given.

Test

tube #

Concentration of

Standard BSA

(mg/mL)

Absorbance

1 Blank Blank

2 0.002 0.0036

3 0.004 0.0040

4 0.006 0.0013

5 0.008 0.0044

6 0.01 0.0034

7 - 0.0058

8 - 0.0038

9 - 0.0051

Table2. Bradford Assay. Bold values are

derived from data given.

In these assays, one thing should

be always taken in mind. A certain

concentration of the desired protein was

already established before any assays are

conducted. In this experiment, 1% egg

albumin and 0.01% casein was used.

This means than every 100mL of

solvent, there is 1g or 1000mg of egg

albumin and in every 100mL of the

solvent, there is 0.01g or 10mg casein. It

is then to be considered that if the

extracted protein is pure and only 1mL

of solution can be added into a cuvette,

the Warburg-Christian Method should

obtain a 10 mg/mL value of

concentration of the egg albumin and 0.1

mg/mL value of concentration for

casein.

Warburg-Christian Method uses

the absorption of tryptophan and

tyrosine residues in 280 nm light. The

absorbance of the tyrosine and

tryptophan residues is directly related to

the protein concentration. Aside from

this, Warburg-Christian Method uses

absorbance at 260 nm light to further

correct the protein concentration if there

are existing nucleic acid impurities.

Despite this incredible method, results

obtained from data given for Warburg-

Christian Method (See Table1) were far

from the expected protein concentration.

The expected protein concentration for

egg albumin was 10mg/mL but the data

gathered for the concentration of

proteins is only in the range of

0.4mg/mL to 0.5mg/mL. To achieve

such minute concentration, the egg

albumin solution should have been

diluted up to 0.05% (w/v) egg albumin

solution considering the obtained

concentration and the concentration of

nucleic acid found in the solution. (To

get % nucleic acid, see Appendix 1).

Only dilution will explain such event if

parameters are correct about the proteins

purity and the presence of nucleic acids.

Unlike the egg albumin solution,

the casein should have a concentration of

0.1mg/mL in the Warburg-Christian

Method however the protein

concentration obtained was greater than

the expected concentration. No dilution

can explain the increase of concentration

value from expected value. Instead, an

error in making the solution will explain

the event. It is given that a 0.01% (w/v)

casein solution is expected to have a

concentration value near 0.1 mg/mL and

for it to gain an increase in concentration

in the data, a casein solution of higher

concentration might have been the

solution used for this experiment or there

have been an error in dilution that did

not achieve the 0.01% (w/v) instead a

solution of higher weight/volume

percent was prepared. Whatever the

case, a certain error was made for the

casein solution to obtain higher protein

concentration than expected especially if

there is only about 3% nucleic acid (See

Appendix 1 for the acquisition of %

nucleic acid) in the solution.

The other assay for protein

concentration determination that is used

in this experiment is Bradford Assay.

Bradford Assay uses Bradford Reagent

that is mainly composed of Coomassie

Brilliant Blue G250. The protein binds

with the Coomassie Brilliant Blue G250

that makes a shift of the wavelength of

maximum absorption from 465 nm to

595 nm. The more proteins bound to the

Coomassie Brilliant Blue G250, the

greater the shift of the wavelength to

higher value. Using this assay, the

absorbance of different concentration of

a standard solution is plotted to form a

calibration curve. On Table2, the data

given for the results of the absorbance of

the spectrophotometer for the Bradford

Assay was the only result that is given.

The protein concentration of the

standard BSA solution in mg/mL was

derived from the methodology.

Using the values in Table2, the

calibration curve for the standard BSA

solution, with the absorbance at the y-

axis and the protein concentration on the

x-axis, will look like this:

Fig.1 Calibration Curve for Standard BSA using

the values from Table2.

Just by a glance at this curve, there is

definitely something erroneous.

Calibration curve is expected to rise as

absorbance also increases as protein

concentration also increases. An error

therefore occurred while obtaining the

data. The error may have come from

mislabeling the solution with different

concentration or the producing a solution

wrong concentrations.

If the error comes from the

production of solution of wrong

concentration, there is a minimal chance

of manipulating the data to form a curve.

Aside from that, the results were just

made up and given not necessarily

obtained in an experiment. If the error

however is made from mislabeling, a

data can be manipulated in such a way

that the concentration is paired with the

absorbance via their values. If such

happened, a new table for data can be

formed.

Test

tube #

Concentration of

Standard BSA

(mg/mL)

Absorbance

1 Blank Blank

2 0.002 0.0013

3 0.004 0.0034

4 0.006 0.0036

5 0.008 0.0040

6 0.01 0.0044

Table3. Manipulated Data from Table3

Using this new table, a new calibration

curve may be produced. The

manipulated calibration curve will look

like this:

Fig.2 Calibration Curve for Standard BSA using

the values from Table3.

The new calibration curve, as seen in

Fig2, from the manipulated data from

Table3 looked like a line more than that

of Fig.1. By using the manipulated data,

a positive slope can be achieved. The

slope of the calibration curve using the

data of Table3 is 0.34. This would mean

than the absorbance of the solution is

equal to the product of the slope and the

protein concentration. Since Table2

contains the absorbance of the samples,

protein concentration can be computed

by getting the quotient of the absorbance

over the slope. Using this, the protein

concentration of the sample will now be

0.017 mg/mL for test tube #7, 0.011

mg/mL for test tube #8 and 0.015 for test

tube #9. Since no protein was specified

in this part of the determination of

protein concentration, it is safe to say

that any of egg albumin or milk casein

was used and the computation of a low

protein concentration suggests that the

solution is diluted even more.

There are still different ways for

determining the protein concentration

like Biuret Assay, Lowry Assay, and

BCA Protein Assay. Biuret Assay uses

the alkaline copper sulfate that forms a

purple complex when is bound to

peptide bonds. The more purple complex

is formed in a solution, the more

concentrated the protein in a solution.

The Lowry Assay is just like the Biuret

Assay just that a second reagent, Folin-

Ciocalteu, was needed to make further

color development and thus

determination. BCA Protein Assay is

also like the Biuret and Lowry Assay.

BCA Protein Assay, however,

bicinchoninic acid and 1% sodium

dodecyl sulfate.

All these assays also require the

use of spectrophotometer. The most used

assays used however are the ones that

were analyzed, Bradford Assay and

Warburg-Christian Method. The

Bradford Assay uses a calibration curve

that determines and predicts protein

concentration. The use of calibration

curve however also applies in Biuret,

Lowry and BCA Protein Assays. Only

Warburg-Christian Method uses an

equation to determine the protein

concentration. The use of an equation to

determine protein concentration is the

advantage of the Warburg-Christian

Method. Aside from that, only Warburg-

Christian Method estimates the

impurities of the protein solution such as

nucleic acids. Though Warburg-

Christian Method estimates that

impurities of the solution, Bradford

Assay uses a calibration curve that not

only determine the protein concentration

but also the prediction of the absorbance

of a solution with a known

concentration.

CONCLUSION

Several methods can be used to

extract and characterize proteins from

certain sources. However, the methods

of extraction have to be effective in such

a way that it will be able to ascertain the

accuracy of concentrations or amounts

of proteins that are being isolated. The

values that the Bradford Assay and

Warburg-Christian method imply a need

for a better technique in the isolation or

extraction of protein samples in order to

produce accurate results.

REFERENCES

Boyer, Rodney F. “Modern

Experimental Biochemistry Second

Edition”. The Benjamin/Cummings

Publishing Company, Inc. 1993

Buckberry, Lorraine and Teesdale, Paul.

“Essentials of Biological Chemistry”.

John Wiley & Sons Ltd. 2001

Campbell, M. K., & Farrell, S. O.

(2009). Biochemistry, 6th edition. 10

Davis Drive, Belmont, California 94002-

3098, USA: Thomson Brooks/Cole.

Chick H, Martin CJ. The Precipitation of

Egg-Albumin by Ammonium Sulphate.

A Contribution to the Theory of the

"Salting-out" of Proteins. Biochem J.

1913 Jul;7(4):380–

398.“http://www.ncbi.nlm.nih.gov/pmc/

articles/PMC1276484/pdf/biochemj0121

2-0042.pdf”

Lehninger, A. L. Biochemistry, 4th

edition. 444 Park Avenue South, New

York, N.Y. 10016, USA: Worth

Publishers, Inc. .

Stoker, H. S. (2010). General, Organic

and Biological Chemistry, 5th edition.

10 Davis Drive, Belmont, California

94002-3098, USA: Brooks/Cole.

APPENDIX

1.

A280/A260 %Nucleic Acid

1.75 0.00

1.63 0.25

1.52 0.50

1.40 0.75

1.36 1.00

1.30 1.25

1.25 1.50

1.16 2.00

1.09 2.50

1.03 3.00

0.98 3.50

0.94 4.00

0.87 5.00

0.85 5.50

0.82 6.00

0.80 6.50

0.78 7.00

0.77 7.50

0.75 8.00

0.73 9.00

0.71 10.00

0.67 12.00

0.64 14.00

0.62 17.00

0.60 20.00