Protein Purification Protocol of the Glycogen Phosphorylase - Term Paper Example

Protein Purification Protocol of The Glycogen Phosphorylase, Muscle form (Human) Name Institutional affiliation Protein Purification Protocol of the Glycogen Phosphorylase, Muscle Form (Human) Introduction Glycogen is one of the most abundant proteins in skeletal muscles consisting of about 4% in total. It important for the utilization of glycogen reserves in the body muscles. Glycogen is a large highly branched polymer of glucose molecules. Most are joined by alpha 1, 4 to make straight chains, and alpha 1, 6 linkages occur every 8 to 12 glucose residues, to make branch points. There are two types of linkage found between the glucose molecules. However, excess Glycogen phosphorylase is usually made of a cofactor pyridoxal phosphate strongly bound to it; this can be easily utilized to measure its degradation. The main sore of glycogen in the human body are found in skeletal muscles and the liver, where they serve as a fuel reserve for the synthesis of energy known as Adenosine Tryphosphate (ATP) during the contraction process of muscles as well as in the liver. Basically glycogen is used to maintain the blood glucose level at equilibrium especially during the early stages of fasting as stated by Pergamon (1990). The Function or Role of Glycogen Phosphoprylase in Human Beings Excess dietary of glucose is usually stored as glycogen in the body muscles. Glucose can be rapidly ad easily mobilized from glycogen when the need arises; for instance, between meals or during exercise. A constant sufficient supply is very crucial for life because it is the main fuel of the brain and the only source of energy that can be used by cells that lack mitochondria or by the frequently contracting skeletal muscles in the process of anaerobic glycolysis. Glycogen is therefore an excellent short-term storage material that can provide energy whenever required with an immediate effect. However, glycogen phosphorylase cleaves the alpha 1,4 bonds between glycosyl residues at the non-reducing ends of the glycogen chains thus producing glucose 1-phosphate. The protein requires pyridoxyl phosphate as a coenzyme to activate the reaction as proposed by Storey (2004). Glycogen is used as an energy reserve. In the brain, it protects against hyp[oglycemia, since this organ functions constantly, while dietary intake of carbohydrates is intermittent. For muscles glycogen provides a more immediate and, and a more abundant source of glucose-phosphate than does blood sugar. It thereby provides the precursors for ATP production that enable more rapid and more extensive muscle action fro limited periods of required activity. It is therefore essential that cleavage of such stored glycogen molecules not occur steadily, since this would deplete the glycogen, or best lead to a futile cycle as glycogen consumption balanced the synthesis of new glycogen as proposed by (Newshome and Leech, 2009). However, muscle store glycogen, but it is not able to convert the Glc-1-P produced to free glucose, and uses the stored glycogen exclusively in the muscles for muscle action. When muscle is resting, it can obtain adequate energy from the use of fatty acids, and glycogen consumption is reserved for active work. Under conditions of active work the muscle phosphorylase is then mainly activated by phosphorylation, in response to a hormone signal, and is also activated by AMP binding at the A site. Glucose itself remains the most effective inhibitor. In skeletal muscles, there are two forms of phosporylase, which are referred to as phosphorylase b (requires AMP for activity) and phosphorylase a (active independent of AMP) interconversion of these two enzyme forms takes place according to the following equations: 2phosphorylase b +4ATP------phosphorylase a + 4 ADP Phosphorylase a + 4 Water--------2 phosphorylase b + 4 Pi Phosphorylase a (phosphorylated form) is considered being the most physiologically active than phosphorylase b (nonphosphorylated form). The predominant form of phosphorylase present at any given time depends upon the relative rates of reaction from the above two equations. These reactions in turn appear to be under hormonal or nervous control. Phosphorylase a formation is promoted by epinephrine by a mechanism involving production of adenosine 3’, 5’-phosphate commonly referred to as the cyclic AMP as well as the activation of phosphorylase b kinase in the Krebs cycle (Traut, 2007). Structure and Properties of Glycogen Phosphorylase A number of pertinent features relating to the structure of human skeletal muscle phosphorylase b and a are mentioned below. Phosphorylase b with a molecular weight (M.W) of 250,000 is made up of two presumably identical subunits of M.W. 125,000. Phosphorylase a has a M.W of 500,000 and contains four of the subunits. In the phosphorylase b to phosphorylase a reactgion, phosphate is usually transferred from ATP to a specific seryl residue in the phosphorylase subunit.phosphorylase a can be looked upon as a phosphoprotein containing four phosphate groups. Phosphorylase b is inactive in the absence of the cofactor AMP, but active in its presence; phosphorylase a does not require AMP but is usually stimulated somewhat by the nucleotide. However, depending on the pH and temperature, phosphorylase a may also be tetramatic, but it has been concluded that the dissociation and association properties do not contribute significantly to the change in enzyme activity as proposed by (Nelson, 2008). The results of properties of glycogen phosphorylase can be summarized in the table below. Property Glycogen Phosphorylase b Glycogen Phosphorylase a Molecular weight (M.W) 250,000 500,000 subunits 2 4 Serine-P 0 4 Stability More stable as a tetramer Not stable as a dimer Activity (-AMP) 0 60-100 Activity (+AMP) 60-100 100 Pyridoxal-P 2 4 Protein Purification Protocol Starting Material and Cell Lysis The initial procedure in the isolation of protein, a protein complex, or a subcellular organelle is the preparation of an extract that contains the required component in a soluble form as suggested by the American Chemical Society (2008). Indeed, when undertaking a proteomic study, the production of a suitable cellular extract is essential. Further separation of subcellular fractions depends on the ability to rupture the animal tissues in such a manner that the organelle or macromolecule of interest can be purified in a high yield, fre from contaminations and in an active form. The homogenization technique employed should, therefore, stress the cells sufficiently enough to cause the surface plasma membrane to rapture, thus releasing the cytosol; however, it should not cause extensive damage to the subcellular structures, organelles, and membrane vesicles. The extraction of proteins from animal tissues is relatively straightforward, as animal cells are enclosed only by a surface plasma membrane that is only weakly held by the cytoskeleton. They are relatively fragile compared to rigid cell walls of many bacteria and all plants and are thus susceptible to shear forces. Animal tissues can be crudely divided into soft muscle like liver and kidney or hard muscle like skeletal and cardiac. Reasonably gentle mechanical forces such as those produced by liquid shear may disrupt the soft tissues, whereas the hard tissues require strong mechanical shear forces provided by blenders and mincers. The homogenate produced by these disrupt methods is then centrifuged in order to remove the remaining cell debris (Roe, 2001). Materials Needed The preparation of extracts from animal tissues requires normal glassware, equipment and reagents. All glassware should be thoroughly cleaned. If in doubt, clean by immersion in a sulfuric-nitric bath. Apparatus should then be thoroughly rinsed with deionized and distilled water. Reagents should be Analar grade or equivalent. In addition, the following apparatus are required: mixers and blenders, refrigerated centrifuge, and finally centrifuge tubes. Method All equipment and reagents should be prechilled to 0-4 degrees Celsius. Centrifuge should be turned on a head of time and allowed to cool down. 1. First, trim fat, connective tissue (muscle), and blood vessels from the fresh chilled tissue and dice intopieces of a few grams 2. Place the tissue in a precooled blender vessel and add cold extraction buffer 3. Homogenize at full speed for 1-3 minutes depending on the toughness of the tissue 4. Remove cell debris and other particulate matter from the homogenate by centrifuging at 4degrees Celsius. 5. Decant the supernatant carefully, avoiding disruption the sedimented material, through a double layer of cheesecloth or muslin. This will remove the fatty material that has floated to the top. Alternatively, the supernatant can as well be filtered via the plug of lass wool placed in a filter funnel. Before further fractionating is undertaken, additional clarification steps may be required in readiness for purification. Protein Purification Isolation from other proteins Fractional Precipitation for Bulk Proteins (size dependent) The solubility of a particular protein in aqueous solution depends on the solvent composition and on the PH; hence, variation in these parameters provide a way purifying proteins by fractional precipitation. The method involves addition of salt (salting out) as well as organic solvents. The addition of high concentrations of salt to a protein solution causes precipitation largely by removing water of salvation from hydrophobic patches on the protein’s surface, thus allowing these patches to interact with resulting aggregation. For a pure protein, the relationship between solubility and the ionic strength is given by the formula: Logs=beta-k {(1/2)}, whereby; beta and k, are constants for a particular protein at a particular PH and temperature. The logic behind this step is that the protein will precipitate over a range of ionic strength values and that different proteins will precipitate alone. With large scale purifications in particular, this is important in reducing the problem to a manageable scale. Hence fractional precipitation by salt is almost invariably used at an early stage of a purification procedure (cutler, 2004). Affinity chromatography Affinity chromatography is a type of adsorption chromatography in which the molecule to be purified is specially and reversibly adsorbed by a complementary binding substance called the ligand that is immobilized on the an insoluble support called the matrix. Purification by this technique can be of the order 100-1000 fold, and recoveries of the bioactive material are genrally very high. Affinity chromatography involves the formation of a reversible complex between the target protein and the ligand. It can, in principle, be applied to a wide variety of macromolecule-ligand systems. However, during the purification of proteins, the target protein from the crude extract interacts with the immobilized ligand and remains bound to it. The other constitutents of the crude extract are dremoved. In some cases, a spencer arm is introduced between the legand and the matrix to improve binding. Desorption of the bound target molecule is generally accomplished by a nonspecific or a specific elution method to provide a ponder nature of affinity interaction. For this case of protein A, textile dyes, and metal ions, the choice of the affinity ligands, which have been used in the affinity chromatography, and other affinity-based approaches, for the understanding of the affinity interaction of protein purification, a molecular pair level of protein is used. During this molecular recognition process, molecules are supposed to show adequate binding because of the biochemical affinity. Conceptually, this so called biochemical affinity was accepted as a fait accompli of nature and simply exploited in an in vitro situation for its capability to pick out one member of an affinity pair using the other. Ion-exchange Chromatography This method is one of the most widely used forms of column chromatography. It is used in research, analysis, and process-scale purification of proteins. Ion exchange is ideal for initial capture of proteins because of its high capacity, relatively low cost, and its ability to survive rigorous cleaning regimes. This method is also ideal of polishing of partially purified protein materials from after undergoing fractional precipitation stage on account of the high-resolution attainable and the high capacity giving the ability to achieve a high concentration of product. Ion exchange chromatography is widely applicable because the buffer conditions can be adapted to suit a broad range of proteins rather than being applicable to a single functional group of proteins. Ion-exchange chromatography relies on the interaction of charged molecules in the mobile phase with oppositely charged groups coupled to the stationary phase. The charged molecules in a buffer solution come from the buffer component like salt. The charged groups on the protein are provided by different amino acids like lysine, arginine, and histidine (positively charged at a physiological pH), and aspartic acid as well as glutamic acid (negatively charged at the physiological pH). Estimation of Purity The purified protein should be placed in an environment that promotes its stability as soon as possible after elution. Frequently, in the case of pH elution, this involves titration to near neutrality. The material may be placed in a suitable buffer by using buffer exchange techniques such as desalting, ultrafiltration, or dialysis. Treatment of the eluted protein will be largely influenced by subsequent purification steps and the use for which the protein is designed as proposed by Doonan (1996) Assay The performance of the above discussed protein purification techniques are usually determined by comparison of the purity before and after purification by polyacrylamide gel electrophoresis (PAGE). Other techniques of assessing purity include, gel filtration and reverse-phase high-performance liquid chromatography (HPLC). However, specific assays should be used to detect recovery (yield percentage) such as; Western Blotting Techniques or, in the case of enzymes, calculating the activity per mass of total protein using techniques such as ELISA. It is important to analyze the flowthrough to ensure maximum recovery. Several methods exist for analyzing the flowthrough material, such as rechomatography to establish that the capacity of the column has not been exceeded. This, however, will not reflect the inability to bind from denaturation of the ligand protein or inappropriate binding conditions (cutler, 2004 References American Chemical Society (2008). Biochemistry, Volume 47, Issues 26-28. New York: American Chemical Society, print. Colfen, H. (1999). Analytical ultracentrifugation V, Volume 5. New York: Springer, print. Cutler P. (2004). Protein purification protocols. New Jersey: Humana Press, print Doonan S. (1996). Protein purification protocols. New Jersey: Humana Press, print. Nelson J. (2008). Structure and function in cell signaling. London, UK: John Wiley & Sons Newshome E., & Leech A. (2009). Functional Biochemistry in Health and Disease. Oxford: John Wiley and Sons Pergamon (1990). Comparative biochemistry and physiology: Comparative biochemistry, Volume 95, Part 2. New York: Pergamon Press, 1990 Roe S. (2001). Protein purification applications: a practical approach. Oxford: oxford university press. Storey B. K. (2004). Functional metabolism: regulation and adaptation. New Jersey: Wiley-IEEE, print. Traut, T. (2007). Allosteric regulatory enzymes. Texas: Springer, print. Read More