Borras, Alyssa Erika L. Cedeo, Trisha Denise D.

Group #3

Date Due: December 3, 2013 Date Submitted: December 3, 2013

EXPERIMENTS 1 & 2 EXTRACTION AND ISOLATION OF PROTEINS/ PROTEIN QUANTITATION THROUGH SPECTROPHOTOMETRIC METHODS Abstract This study aims to extract, isolate and quantify proteins such as albumin and casein from natural sources, while at the same time understand the principles governing such processes. Albumin was isolated from egg white solution using ammonium sulfate, a process known as salting out. Casein, on the other hand, was isolated from milk through differential solubility. Quantitation of proteins was achieved by utilizing UV-Vis spectrophotometer. Different assays were conducted to determine the exact concentration of the samples. Extracted albumin was found to have 11.3% concentration; while extracted casein precipitate has 14.4 % concentration. In Warburg-Christian assay, albumin has concentration of 0.430 mg/ml and 0.060 mg/ml for casein. Concentrations obtained in Bradford and Biuret assays showed a trend where the relationship of concentration and absorbance was directly proportional. Errors encountered in the study were determinate in nature. The results of this study can add to the present understanding of extracting, isolating and quantifying proteins.

Results Proteins constitute the major fraction of the mass of all organisms. The extraction, isolation and purification processes are only the first step in the characterization of proteins. Properties of proteins such as solubility must always be considered in order to employ right techniques that will lead to sufficient amount of crude product. Table 1 presents the appearance and properties of crude protein extracts. Table 2 shows the concentration of extracted proteins.
Table 1. Appearance and properties of crude protein extracts

Casein After centrifugation (ppt and sup) After ethanol washing After acetone washing

white ppt/ thick, cloudy supernatant white ppt/ clear supernatant white ppt/ clear supernatant

Table 2. Calculated quantities and concentrations of proteins from crude protein extract

Sample

Mass of Mass Concentration centrifuge of of protein (%) (g) protein (g) 6.64 10.24 10.74 11.30 % 14.40 %

Albumin Casein 6.63

Crude Protein Properties/Appearance Extract Albumin white ppt Ppt after 1st fractionation Ppt after 2nd white ppt fractionation

The determination of the quantity of protein during protein purification is necessary to identify whether the isolation processes are efficient. There are several methods used to identify protein quantity

and concentration. Protein quantitation methods are mostly colorimetric techniques. Most colorimetric protein assay methods can be divided into two groups based on the type of chemistry involved: those involving protein-copper chelation with secondary detection of the reduced copper and those based on protein-dye binding with direct detection of the color change associated with the bound dye. The methods used in the experiment (except for the Warburg-Christian method) are primarily based on protein-dye binding. The following results show the different absorbance and hence, different concentrations of protein extract used. In WarburgChristian method, the crude extracts were directly used for the measurement of absorbance while in Bradford and Biuret Assays, dilutions and addition of reagent were done prior to the measurement of absorbance.
Table 3. Absorbance values of albumin and casein using Warburg-Christian Assay

Table 5. Results for the Biuret Assay.

Protein Albumin Concn (% w/v) Casein Concn (% w/v) 3

Increasing Intensity (Test tube#) 2 1 4

Calibration curves were prepared from the data of test tubes 1to 6 which served as the standard curves.

Standard Calibration Curve

BSA Concentration 1 0.5 0 0 0.005 0.01 0.015 y = 22.071x + 0.6188 Absorbance R = 0.7648

A280 A260 A280/ A260 Average Ratio Protein concentration (mg/mL)

Protein Extract Albumin Casein T1 T2 T1 T2 0.411 0.417 0.085 0.074 0.278 0.279 0.089 0.077 1.478 1.495 0.955 0.960 1.487 0.958 0.430 0.060

Figure 1. Calibration curve for Bradford Assay

Discussion of Results In this experiment, albumin and casein were extracted from egg white and milk, respectively due to the abundance of the target proteins from the said sources. The purification procedure takes into account the physical and chemical properties of the protein such as solubility, polarity and temperature needed to obtain maximum recovery. Suitable purification methods are determined by the activity of the target protein to different solvents and treatments, and the contaminating compounds present together with the target protein. 1

Table 4. Absorbance values for each BSA and crude protein extract using Bradford Assay.

Standard BSA
Test tube no. Absorbanc e 1 0.56 7 2 0.70 1 3 0.69 9 4 0.80 8 5 0.81 3 6 0.78 7

Sample test tube no. Absorbanc e

Albumin 7 8

Casein 10 11

12

0.76 1

0.61 0

0.69 2

0.65 5

0.66 5

0.62 0

Purification started by the disruption of the cell membrane of the source through homogenization to isolate the target proteins. Homogenization by mechanical disruption as in the case of egg white was done by stirring and pressing of the egg white during filtration. 2 Once the membranes were disrupted, the target proteins were isolated by centrifugation and fractional precipitation. Centrifugation is the process in which particles in a solution is separated accordingly depending on size, shape and density. In this experiment, centrifugation was done to separate the target proteins, nucleic acids and other unbroken cells from the broken ones. On the other hand, fractional precipitation involves the separation of particles from the solution depending on its solubility. In this experiment, fractional precipitation may be observed when pH was lowered, organic solvents and neutral salts were used and when there was a need for ice bath and refrigeration. Isoelectric precipitation is the adjustment of the pH of a solution as to reach its isoelectric point, as may be seen in the extraction of casein in milk using HCl. Proteins, having both positive and negative charges due to the amino and carboxyl groups present may then have a neutral net charge. Isoelectric point is the point in which the net charge of the protein is equivalent to zero. At this point, because of the absence of a charge, a decrease in the protein-water molecule interaction causing for the solubility to be lowered will result to an increase in protein-protein molecule interaction thereby forming a precipitate. The same concept may be observed when organic solvents miscible with water, such as ethanol and acetone, were used. Such solvents have a low dielectric constant which further lowers that of the water which decreases its solvating power by increasing exposing more hydrophobic sites that promotes hydrophobic interactions. 3 Proteins also have different solubility when neutral salts are added. They tend to be insoluble to concentrated salt solutions, as may be seen when

saturated (NH4)2SO4 was used in the extraction of albumin from egg whites. This is because of the fact that when the concentrated salt solution was added, the ions tend to attract the water molecules blocking the hydrophobic sites of the protein. Once these sites are exposed, protein molecules interact with one another causing in aggregation and therefore forming insoluble precipitates. This is called the salting out effect. 2 On the other hand, when dilute concentrations of salt was used as in the use of 0.01 M NaOH and 0.9% NaCl, proteins tend to be soluble because of the weakening of the attractive forces between proteins, called salting in effect. Lastly, proteins, when exposed to high temperatures, denature which disrupts the natural conformation of the proteins affecting its function. Therefore, to avoid denaturation, the process should be done at lower working temperatures, and refrigeration was needed for the isolated proteins. However, in plants and microorganisms, simple mechanical disruption of cell membrane is not enough due to the presence of cell wall and other compounds such as phenols and proteinases which protects further the contents of the cell and decreases the activity of the target protein. To address these problems, enzymatic lysis using lysozyme and extraction buffers is applied to digest and stabilize the membrane. Nevertheless, certain part of plants that are rich in specific enzymes and thus, used primarily for extraction such as the seed, tuber and tap roots also known as storage organs. On the other hand, a direct approach is applied to animal tissues due to weak membrane holding the target proteins. The purification method for animal tissues then depends on source, whether it is a soft or hard muscle, and to the complexity of the target protein, extracellular matrix or membrane bound. Extracellular matrix proteins require more extensive extraction with the use of chemical hydrolysis and/or proteolytic cleavage because of low solubility than the membrane bound. 4, 5

After extraction, the proteins were subjected to quantitation through spectrophotometry, employing three different methods: Warburg-Christian Assay, Bradford Assay and Biuret Assay. Warburg-Christian Assay involves the direct measurement of UV absorbance of amino acids such as Tyrosine and Tryptophan at 280 nm since most proteins also exhibit absorbance at the said wavelength. However, since proteins may contain different amount of amino acids, this method is not suitable for quantitative determination of concentrations of protein mixture. Even though, this technique, being non-destructive, fast and easy, also provides correction for contaminants such as nucleic acids which commonly absorbs at 260 nm. On the other hand, both the Bradford and Biuret Assay are colorimetric methods which employ the binding of Coomassie Brilliant Blue dye and Cu2+ respectively, to the protein. While the Bradford Assay causes a shift in the absorption wavelength of the dye from 465 to 595 nm due to its binding with the protein, the Biuret Assay forms a violet solution due to the complexation of the protein and Cu2+ with absorbance that can be measured at 540 nm. 6 For the determination of protein concentration through Warburg-Christian Assay, the calculated protein concentration for albumin and casein were 0.430 mg/mL and 0.060 mg/mL, respectively. Using Bradford Assay, the Standard BSA absorbance values ranged from 0.567 to 0.813. For the samples, the absorbance values for albumin ranged from 0.610 to 0.761 while for casein, the values ranged from 0.620 to 0.665. On the other hand, Biuret assay was able to qualitatively show the trend in increasing protein concentration of albumin and casein through the observable increasing intensity of violet color per test tube. For albumin, test tube 3 showed the highest intensity followed by test tubes 2, 1, 4 and 5. Meanwhile, test tube 2 exhibited the highest intensity for casein, followed by test tubes 3, 1, 4 and 5.

Other methods for protein quantification include the Lowry method and Bicinchoninic acid (BCA) method, both colorimetric methods takes advantage of the reducing power of amino acids such as Tyrosine and Tryptophan, at alkaline solutions to reduce Cu2+ to Cu+ which would later bind to the reagent. In Lowry method, the Cu+ binds with the Folin-Ciocalteu reagent which is a reduced phosphomolybdate-phosphotungstate solution to produce a blue color solution to be measured at 660 nm. This process, however, destroys the target protein. Meanwhile, in BCA Assay, the Cu+ binds with the BCA to produce a violet colored solution measured at 562 nm. Though the color yield varies between proteins due to the number of reducing amino acids, this method is useful in determining the changes in protein content as it can be used in varying temperatures. This method should be accompanied with the use of buffer to eliminate the error brought about by non-protein reducing compounds. 3 Most protein quantification methods are neither straightforward nor exact due to the variations in the properties of target protein. Methods present cannot address fully each problem such as the presence and amount of amino acids in each protein, and the presence of contaminating compounds that has not been full separated from the target protein. Variations in the result may be due to personal errors in the preparation of solutions used in the experiment and in the preparation of the sample during the use of the spectrophotometer, as fingerprints or bubbles may be present in the cuvette, blocking the path of the light. Differences as well in the degree of mechanical disruption (stirring etc) may cause the lower values of extracted proteins. REFERENCES: [1] Janson H.C. & Ryden L. 1998. Protein Purification 2nd Edition. John Wiley & Son, Inc. Canada. pp.5-7

[2] Campbell, M.K. & Farrell, S. O. 2012. Biochemistry 7th Edition. Brooks/Cole, Cengage Learning. USA. pp.117-118 [3] Switzer, R. L. & Garrity, L. F. 1999. Experimental Biochemistry. W.H. Freeman and Company. USA. pp.91-94 [4] Fido, R. J. et al. 2004. Protein Extraction From Plant Tissues, Methods in Molecular Biology, vol.244: Protein Purification Protocols 2nd Edition Ed by P. Cutler. Humana Press Inc. USA. [5] Shekel, J. M. 2004. Preparation of Extracts From Animal Tissues, Methods in Molecular Biology, vol.244: Protein Purification Protocols 2nd Edition Ed by P. Cutler. Humana Press Inc. USA. [6] 2013. Biochemistry Laboratory Manual. Biochemistry Academic Group, Institute of Chemistry, UP Diliman. Philippines [7] Protein Analysis- Determination of Protein Concentration. Retrieved from <http://public.callutheran.edu/~revie/biochemistry/Pr otein-analysis-lab.pdf> [8] 2009. Comparison of Protein Assays. Retrieved from < http://www.colby.edu/chemistry/CH367/laboratory/ex pt2.pdf> [9] Sattayasai, N. Protein Purification. Retrieved from <http://cdn.intechopen.com/pdfs/28765/InTechProtein_purification.pdf>