Extraction of Proteins Essay

Biochemistry Laboratory (BCM 362L), Experiment No. 3, © February 14, 2006 3nd Quarter A. Y. 2005-2006 Extraction and Characterization of Proteins Mr. *****1, *****2, *****2 1 Professor, School of CHE-Chm, Mapua Institute of Technology 2 Student, BCM 362L/ A31, School of CHE-Chm, Mapua Institute of Technology ____________________________________________________________ __________________________ ABSTRACT Invertase, Albumin and Casein were extracted from yeast, egg and milk respectively using solvent extraction, salting out and isoelectric precipitation.

Benedict’ test was performed to determine the presence of reducing sugars in the prepared solution. Concentrations of albumin and casein were determined by two methods – Warburg-Christian method and Bradford Assay. By using the Warburg-Christian method, the concentration of casein is 0. 3329 mg/mL. Different concentrations for different absorbance was computed using Beer’s law for the Bradford assay method, a linear plot was calibrated. Keywords: extraction, assay, isoelectric, calibrated. ____________________________________________________________ __________________________

We will write a custom essay sample on
Extraction of Proteins Essay
or any similar topic only for you
Order now

INTRODUCTION Proteins are the most diverse and abundant macromolecules within cells. Their functions range from the enzymes that carry out the numerous metabolic processes of the cell to structural components that gives cells structure and organization. One of the most useful parameters used in the study of proteins in solution is the concentration of the protein and. It is necessary for biochemists to estimate the concentration of proteins in a solution of interest in order to quantitatively determine the activity of the protein. In one part of the experiment, Benedict’s test was used.

Here, monosaccharides and disaccharides can be detected because of their free aldehyde groups, thus, testing positive for the Benedict’s test. Such sugars act as a reducing agent, and is called a reducing sugar. By mixing the sugar solution with the Benedict’s solution and adding heat, an oxidation-reduction reaction will occur. The sugar will oxidize, gaining an oxygen atom, and the Benedict’s reagent will reduce, loosing an oxygen atom. There are two types of spectrophotometric methods for determining protein concentration, which will be used for albumin and casein.

The first method is called the Warburg-Christian method. In this method the protein concentration is estimated by making a direct absorption measurement of a solution in the UV range. The advantage of this method is that it is direct and it is nondestructive. The method also has disadvantages. First, the absorption at 280 nm will vary from protein to protein depending on the content of tyrosine and tryptophan.. Therefore, absolute quantitation of protein concentration by this method can only be determined for pure preparations of protein. Second, any substance that absorbs at 280 nm will interfere with the reading.

However, the method can be quite useful as a rapid and nondestructive method to compare the relative concentrations of numerous protein solutions, such as the fractions generated during protein purification by column chromatography. The sensitivity of the assay varies with the tyrosine and tryptophan content of the protein solution, but is in the range of 1 mg protein/ml to 1 mg protein/ml. The second method to determine protein concentration is the protein dye-binding assay or Bradford assay. The dye, Coomasie brilliant blue, is a relatively hydrophobic substance that binds avidly to the hydrophobic regions of proteins.

The change in absorption can be used to quantitate indirectly the amount of protein in the solution. The color change can be quantified with a spectrophotometer. There are two major advantages to the Bradford assay: 1) the ease of performance and 2) few interfering substances. The only interfering substances are high concentrations of detergents that disrupt the binding of the dye to protein because of their amphipathic nature. The sensitivity of the Bradford assay is in the range of 1 mg to 100 mg of protein. 3, 4 METHODOLOGY The proteins invertase, albumin and casein were prepared from yeast, egg and milk respectively for analysis.

Invertase from Yeast A clean mortar and pestle was prepared and to it 10 g of baker’s yeast with 5 g of sand was grinded until the fine powder was obtained. 10 mL of hexane was added to this fine powder. 30 mL of water in 3 mL portions was added and the grinding was continued for 15 minutes. A cell free extract was prepared by filtering the grounded yeast through cheesecloth. The filtrate was centrifuged at 6000 rpm for 5 minutes and the sediment was discarded. The centrifugation step was repeated to get the clear supernatant. The supernatant was poured into a 50-mL beaker and 200 mL of 95% ethanol was slowly added.

It was set in an ice bath until the precipitation had occurred. The resulting suspension was centrifuged at 3000 rpm for 3 minutes. The supernatant was discarded. The invertase crude extract will be used for the analysis. Four test tubes were set according to the table below: |Volume of solutions (mL) | |Test |0. 3 M |H20 |Buffer |2% Glucose|2% Fructose| |tube # |Sucrose | | | | | |1 |6. 0 |4. 0 |6. 0 |- |- | |2 |6. 0 |8. |6. 0 |- |- | |3 |- |8. 0 |6. 0 |1. 0 |1. 0 | |4 |- |10. 0 |6. 0 |- |- | The test tubes were placed in a water bath which was maintained at 37? C and equilibrated for 10 minutes. 4 mL of invertase extract was added to test tubes 1, 3, 4. The tubes were kept in the water bath for 6 minutes more. The reaction was stopped by adding 2 mL of 10% NaOH to the test tubes. The presence of reducing sugars was tested using Benedict’s reagent. Albumin from Egg 0 mL of egg white was measured and was placed in a beaker. The egg white was stirred with a stirring rod and 3 mL of 1. 0 M HOAc was added. The resulting mixture was filtered through the cheesecloth by stirring vigorously with a glass rod to speed up the passage of the filtrate through the cheesecloth and was squeezed to break the membranes. The filtrate was collected in a 250-mL beaker. An equal volume of saturated (NH4)2SO4 solution was added to the filtrate. The mixture was allowed to stand for 30 minutes. The mixture was centrifuged and the precipitate was discarded.

The supernatant was transferred into a 250-mL Erlenmeyer flask. (NH4)2SO4 buffer was added to the clear yellow supernatant while it was continuously stirring until turbidity persists. The mixture was then cooled for two days to allow precipitation of albumin. The mixture was centrifuged and the resulting supernatant was discarded while the precipitate was collected. The crude precipitate was weighed. A 10% solution of albumin in 0. 9% NaCl was prepared. Casein from Milk 25 mL of whole milk was prepared by diluting it with 75 mL of skim milk. The prepared milk was heated to 40?

C while constantly stirring. 0. 1 M HCl was added drop-wise over a period of 10 minutes until a flocculent precipitate forms. The curd was allowed to settle and the whey was decanted. The curd was then washed by resuspending it in 15 mL of water while stirring vigorously. RESULTS AND DISCUSSION Benedict’s test Yeast (Saccharomyces cerevisiae) invertase is essentially an intracellular enzyme and disruption of cell membranes is necessary for its extraction. The crude extract, invertase, prepared by autolysis of dried baker’s yeast gives satisfactory activity with no interfering activities.

The yeast enzyme invertase is a b-fructofuranosidase that can hydrolyze sucrose which is D-glucopyranosyl (1(2)-b-D-fructofuranoside into glucose and fructose. Generally, invertase acts as a catalyst to convert sucrose into glucose and fructose. [pic] A simple test using Benedict’s reagent containing a solution of copper sulfate, sodium hydroxide, and tartaric acid was performed to indicate the presence of reduced sugar in the test tubes. |Test tube # |Description | |1 |(+) – reddish brown | |2 |(+) – light reddish brown | 3 |(+) – dark reddish brown | |4 |(-) | Benedict’s reagent when heated with glucose or fructose reduces the blue copper (II) ion to form a positive result by the presence of brick red solution of copper (I) oxide. Positive results (red solutions) were obtained for test tubes 1, 2 and 3. For test tube 1 and 2 a positive result was obtained. Test tube 1 was found to be darker than test tube 2 because test tube 1 has a higher concentration of glucose and fructose due to the presence of the catalyst invertase which speed up the reaction of sucrose.

Test tube 2 does not contain invertase and hence a slow reaction is present which means less amounts of glucose and fructose were formed during the time. Test tube 3 was proven positive and the darkest of them all because aside from the presence of invertase, the solution already contains glucose and fructose and thus making it highly concentrated. The result for test tube 4 was negative because of the absence of sucrose, a non-reducing sugar which does not react with Benedict’s reagent Warburg-Christian method

Proteins exhibit a strong absorption at 280 nm due to the absorption of the aromatic rings of their intrinsic tyrosine and tryptophan amino acid residues. This includes nucleic acids that are one of the most common contaminants of protein preparations. The data obtained is tabulated below. Albumin was not analyzed due to spectrometer malfunction. |Protein |A280 |A260 |A280 / A260 |Protein | |Extract | | | |Concentration | | | | | |(mg/mL) | |Casein |0. 4171 |0. 4127 |1. 01 |0. 3329 |

By using the table of the absorbance of nucleic acid, the casein concentration can be computed by linear regression. Bradford Assay The Bradford assay is a very popular protein assay method because it is simple, rapid, inexpensive and sensitive. The Bradford assay works by the action of Coomassie brilliant blue G-250 dye (CBBG). This dye specifically binds to proteins at arginine, tryptophan, tyrosine, histidine and phenylalanine residues. CBBG binds to these residues in the anionic form, which has an absorbance maximum at 595 nm (blue). The free dye in solution is in the cationic form, which has an absorbance maximum at 470 nm (red).

The assay is monitored at 595 nm in a spectrophotometer, and thus measures the CBBG (see figure) complex with the protein. [pic] One crucial part of this assay is the buffer blank. Since the assay responds non-linearly it is highly important to lock down the zero point. Because this point is so important to the curve fit, it is highly recommended that at least two buffer blanks be performed. The absorbance and concentrations are tabulated below. For Albumin: |Test Tube # |A595 |Protein Concentration | | | |(mg/mL) | |1 |0. 59 |0. 024 | |2 |0. 884 |0. 0359 | |3 |0. 0734 |0. 004 | |4 |0. 0786 |0. 0013 | |5 |0. 0877 |0. 0044 | |6 |0. 093 |0. 0046 | |7 |0. 0643 |0. 0034 | |8 |0. 0664 |0. 0035 | |9 |0. 0644 |0. 0034 | |10 |0. 0628 |0. 034 | |11 |0. 0612 |0. 003 | |12 |0. 0619 |0. 0033 | For Casein: |Test Tube # |A595 |Protein Concentration | | | |(mg/mL) | |7 |0. 0808 |0. 0041 | |8 |0. 0821 |0. 0038 | |9 |0. 0975 |0. 0048 | |10 |0. 1033 |0. 0051 | |11 |0. 1244 |0. 059 | |12 |0. 1190 |0. 0052 | The concentrations can be calculated using Beer’s Law. Casein has a higher absorbance reading than albumin which means that casein has more proteins that binds with CBBG. CONCLUSION The redder the solution in the Benedict’s test, the more concentrated is the glucose and fructose in the solution. The enzyme invertase helps catalyzes the solution. The Warburg-Christian method was used to determine the concentration of Casein which was 0. 3329 mg/mL. Proteins are absorbed at 280 nm while nucleic acid absorbs at the 260 nm region.

Coomassie brilliant blue dye reacts with certain amino acids which affects the absorbance reading. Absorbance is directly proportional to concentration. LITERATURE CITED 1. Biochemistry, Garrett and Grisham, 3rd Edition 2. © 1993-2003 Microsoft Corporation. All rights reserved. 3. http://tecn. rutgers. edu/bio301s/Lab%203-%20protein%20diagram. htm 4. http://jchemed. chem. wisc. edu/JCESoft/cca/cca5/MAIN/1ORGANIC/ORG18/TRAM18/B/MENU. HTM 5. http://www. essaysample. com/essay/001114. html 6. http://www. lsbu. ac. uk/biology/enzyme/practical1. html


Hi there, would you like to get such a paper? How about receiving a customized one? Check it out