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 proteindye 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.

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.

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 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 Concentration

of

Protein

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.

Casein the Warburg-Christian Method should obtain a 10 mg/mL value of Trial 1 Trial 2 concentration of the egg albumin and 0.1 A280 0.4171 0.4169 mg/mL value of concentration for A260 0.4127 0.4138 casein. A280/A260 1.0107 1.0075 Warburg-Christian Method uses Average 1.0712 1.0712 1.0091 1.0091 absorption of tryptophan and the Ratio tyrosine residues in 280 nm light. The Protein absorbance of the tyrosine and concentration 0.5013 0.3988 0.3329 0.3317 tryptophan residues is directly related to (mg/mL) the protein concentration. Aside from Table1. Warburg-Christian Method. Bold values are derived from data given. this, Warburg-Christian Method uses absorbance at 260 nm light to further Concentration of correct the protein concentration if there Test Standard BSA Absorbance are existing nucleic acid impurities. tube # (mg/mL) Despite this incredible method, results 1 Blank Blank obtained from data given for WarburgChristian Method (See Table1) were far 2 0.0036 0.002 from the expected protein concentration. 3 0.0040 0.004 The expected protein concentration for 4 0.0013 0.006 egg albumin was 10mg/mL but the data 5 0.0044 0.008 gathered for the concentration of 6 0.0034 0.01 proteins is only in the range of 7 0.0058 0.4mg/mL to 0.5mg/mL. To achieve 8 0.0038 such minute concentration, the egg 9 0.0051 albumin solution should have been Table2. Bradford Assay. Bold values are diluted up to 0.05% (w/v) egg albumin derived from data given. solution considering the obtained In these assays, one thing should concentration and the concentration of be always taken in mind. A certain nucleic acid found in the solution. (To concentration of the desired protein was get % nucleic acid, see Appendix 1). already established before any assays are Only dilution will explain such event if conducted. In this experiment, 1% egg parameters are correct about the proteins albumin and 0.01% casein was used. purity and the presence of nucleic acids. This means than every 100mL of Unlike the egg albumin solution, solvent, there is 1g or 1000mg of egg the casein should have a concentration of albumin and in every 100mL of the 0.1mg/mL in the Warburg-Christian solvent, there is 0.01g or 10mg casein. It Method however the protein is then to be considered that if the concentration obtained was greater than extracted protein is pure and only 1mL the expected concentration. No dilution of solution can be added into a cuvette, Albumin Trial 1 Trial 2 0.5786 0.4935 0.5204 0.4817 1.1118 1.0245

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 yaxis 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 # 1 2 3 4 5 6 Concentration of Standard BSA (mg/mL) Blank 0.002 0.004 0.006 0.008 0.01

Absorbance Blank 0.0013 0.0034 0.0036 0.0040 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, FolinCiocalteu, 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 WarburgChristian Method estimates the impurities of the protein solution such as nucleic acids. Though WarburgChristian 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 940023098, 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 1.75 1.63 1.52 1.40 1.36 1.30 1.25 1.16 1.09 1.03 0.98 0.94 0.87 0.85 0.82 0.80 0.78 0.77 0.75 0.73 0.71 0.67 0.64 0.62 0.60 %Nucleic Acid 0.00 0.25 0.50 0.75 1.00 1.25 1.50 2.00 2.50 3.00 3.50 4.00 5.00 5.50 6.00 6.50 7.00 7.50 8.00 9.00 10.00 12.00 14.00 17.00 20.00