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Isolation and Characterization of Proteins

[Isolation of protein and Hydrolysis of intact protein] Riczen Mae Vila, Kathyrene Visco, Rafael Vivar, Jenninah Mae Zafra, and Jeselle Ziganay Group 8 2 G Pharmacy General Biochemistry Laboratory Abstract
A protein is a naturally occurring, unbranched polymer in which the monomer units are amino acids. More specifically a protein is a peptide in which at least 50 amino acids residues are present. Proteins can be classified into two types: fibrous and globular. Fibrous proteins are proteins in which peptide chains are arranged in long strands or sheets. Globular proteins are proteins that tend to fold back on themselves into compact spheroidal shaped units. Globular proteins do not form intermolecular interactions between protein units and are more easily solubilized in water as colloidal suspensions than fibrous proteins are. In this experiment, all the samples are in globular form. The experiment assigned to group 8 is to isolate casein from skimmed milk. Having a sample of 20.0g milk, following the procedures, the group obtained 6.5g of casein. Computing weight of casein obtained over weight of the milk multiply by 100%, the group achieved a weight percentage of 32.5%.

Introduction [1]Proteins is a large molecule composed of one or more chains of amino acids in a specific order determined by the base sequence of nucleotides in the DNA coding for the protein. It is an essential constituent of all cells. [2]Proteins are the most abundant organic molecules found in living cells. They are polymers of amino acids. There are 20 different amino acids found in proteins namely aspartate, glutamate, lysine, arginine, histidine, tyrosine, cysteine, tryptophan, phenylalamine, methionine, serine, threonine, asparagine, glutamine, valine, isoleucine, glycine, alanine, leucine, and proline. There are four points considered in the isolation of an intact protein from its source. These are the Three-dimensional (3D) structure of the protein (fibrous or globular), interactions that keep the native conformation of the protein, acidbase property, and solubility of protein in different solvents. Their solubility can be altered by changing the pH of their environment. Proteins are insoluble and their isoelectric pH, the pH at which the net change is equal to zero. To denature proteins is to disrupt the native conformation. It can be done by subjecting the proteins to extremes of heat and pH, and different denaturing solvents. Denaturing proteins will alter its function, demonstrating a relationship between structure and function. In this experiment, it is intended to isolate casein and albumin from skimmed milk, gluten from wheat flour, and myoglobin from muscle.

Caseins, lactalbumins and lactoglobulins are globular proteins found in milk. Casein exists in milk as calcium caseinate. To isolate casein at its isoelectric pH, an acid is used to adjust the pH to 4.6. Albumins are globular proteins that are soluble in water and in dilute salt solutions. However, albumins can be denatured and coagulated by heat. Gluten is isolated from wheat flour by washing the dough with water. This removes the starch leaving the insoluble material, gluten. Myoglobin and hemoglobin are vital to oxygen transport in vertebrates. The former is present in large concentrations in muscles and is responsible for the red color of the organ. Myoglobin can be isolated by ammonium sulfate precipitation from the buffered muscle extract. Enzymatic, Acidic and Alkaline Hydrolysis of intact protein was also done in this experiment. The 20 amino acids commonly found as hydrolysis products of proteins contain an -carbonyl, an amino group, and a distinctive R group substituted on the -carbon atom. Hydrolysis of the protein and analysis of the product are done to obtain information about their compositions. Hydrolysis can be carried out by treating the protein with acid, alkali or proteolytic enzymes. In acidic hydrolysis, hydrochloric acid will be used. It vaporizes when heated then comes in contact with the protein sample and hydrolyzes the sample. On the other hand, sodium hydroxide will be used on the intact protein for alkaline hydrolysis.

Enzymatic hydrolysis is the test to the presence of proteases. It is also known as peptidases or proteolytic enzyme. These occur naturally in all organisms. They cut peptide bonds of proteins at specific amino acid residues producing peptide fragments and some free amino acids. Currently, there are six classes of peptidases specific to either N- or C-side of the following amino acid residues: serine, threonine, cysteine, aspartate, glutamate, metallopeptidases.

Decant off the supernatant. Describe the appearance of the purified myoglobin residue. Acid hydrolysis of intact protein Add 5mL 6M Hydrochloric acid to 0.5g isolate protein in a hard glass test tube then label. Stopper the tube with cotton and submit to the instructor for autoclaving (15 psi for 5 hours). Take note the appearance of the mixture after autoclaving. Add 10mL distilled water to the mixture then transfer to a 250-mL beaker. Neutralize the mixture with 1M sodium hydroxide for characterization tests and thin layer chromatography. Alkaline hydrolysis of intact protein Add 10 mL 4M sodium hydroxide to 0.5g isolated protein in a hard glass test tube then label. Stopper the tube with cotton and submit to the instructor for autoclaving (15 psi for 5 hours). Take note the appearance of the mixture after autoclaving. Add 10mL distilled water to the mixture then transfer to a 250-mL beaker. Neutralize the mixture with 1M hydrochloric acid for characterization tests and thin layer chromatography. Enzymatic hydrolysis Place 1g/100mL distilled water protein mixture. Mix 10mL of protein mixture and 10mL of saturated protease solution. Alternatively, 0.050g of protease may be added directly to 50mL protein mixture. Add 10mL 0.1M phosphate buffer pH 7.5. Incubate the tube in water bathmaintained at 35-40C (depending on the source of enzyme) for 60 minutes. Alternatively, digestion may be carried out overnight at room temperature. Allow the mixture to cool before using it for tests for qualitative color reaction and separation and identification of amino acids by thin layer chromatography.

Methodology Casein from Skimmed milk Place 20.0g of powdered non-fat milk and 50.0mL of water into a 100-mL beaker. Mix well the solution. Heat the mixture to about 40C. Do not exceed 40C. Monitor the temperature using a thermometer. When the mixture has reached 40C, add 10% acetic acid drop wise. Stir the solution gently after 5 drops. Continue adding acetic acid until pH reaches 4.6. Filter the congealed casein by vacuum or gravity filtration. Set aside the decantate of the isolation of albumin. Dry the casein residue and calculate the weight % of the casein isolated from powdered milk. Albumin from skimmed milk Heat and decantate to 75C for about 5 minutes the decantate from the isolation of casein. After wards, decant off the liquid form the precipitated albumin. Gluten from wheat flour Add enough water to one cup of wheat flour to make a thick dough. Wrap the dough in cheesecloth then place under running water until all starch is removed. Test the washings with I2 solution until a negative result is obtained. The insoluble material is the crude gluten Myoglobin from muscle Place 6.0g of minced beef heart (or steak) and 6mL 70% ammonium sulfate solution in a small beaker pH 7.5 [salting in].Gently stir the mixture for 1 minute to release the myoglobin. Express the dark-red extract into a new beaker using cheesecloth. Centrifuge the extract at 13,000 x g for 5 minutes. Transfer 1.5mL of the supernatant into another centrifuge tube. Add ~0.30-0.35 g of ammonium sulfate crystals ground to fine powder [salting out]. Mix gently until the solid dissolves and avoid frothing. Centrifuge the sample again for at least 5 minutes.

Results *the group is assigned to experiment casein from milk. Other data is recorded by the other groups. Table1. Description of results of isolation of proteins Properties Gluten Casein %w/w 32.5% Albumin Myoglobin Description It is a yellow, sticky claylike form substance White in color, like cream cheese

destabilized or aggregate because the electric charge is decreased to that of the isoelectric point (pH at which there is no net charge becasue there are equal number of positive and negative charges present). The casein micelles disintegrate and the casein (the neutral protein) precipitates because it is no longer polar, with the calcium ions remaining in solution. References [1]http://www.medterms.com/script/main/art.a sp?articlekey=6554 [2]Bayquen Ph.D., Aristea V., et al. Laboratory Manual in ORGANIC CHEMISTRY. Manila: C&E Publishing, Inc. [3]http://homepages.ius.edu/dspurloc/c122/case in.htm

Isoelectric precipitate; from milk Milky yellow curd


Figure1. Computations for weight % of the casein isolated from powdered non-fat milk Conclusion [3]The main protein in milk is casein. Casein is a phosphoprotein which has phosphate groups attached to the hydroxyl groups of some of the amino acids side-chains. Casein exists in milk as a calcium salt, calcium caseinate. It is actually a mixture of three similar proteins, alpha, beta and kappa caseins which form a micelle. Alpha- and betta-casein are both insoluble in water and are solubilized by the micelle surrounding them. The kappa-casein which has a hydrophilic portion is responsible for solubilizing the other two caseins by promoting the formation of and stabilizing the micelles. Calcium caseinate has an isoelectric point of pH 4.6. This means it is insoluble in solutions with a pH less than 4.6. The pH of milk is 6.6, therefore, casein has a negative charge at this pH and is solubilized as a salt. If an acid is added to milk, the negative charges on the outer surface of the casein micelles are neutralized, by protonation of the phosphate groups. The casein micelles are

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