Enzyme Purification Methods PDF

USMR-353 Enzymology Enzyme Purification By Dr. Niranjan P. Patil Extraction Extraction is used to liberate a product of microbial growth from the cells or cellular constituents that served as the enzyme source either by mechanical or non-mechanical means.  Principles an...

USMR-353 Enzymology Enzyme Purification By Dr. Niranjan P. Patil Extraction Extraction is used to liberate a product of microbial growth from the cells or cellular constituents that served as the enzyme source either by mechanical or non-mechanical means.  Principles and methods of enzyme purification: i. Based on molecular size ii. Based on charge iii. Based on solubility differences iv. Based on specific binding property and selective adsorption i) Based on molecular size. This principle includes methods like dialysis, ultra filtration, density gradient or zonal centrifugation and molecular exclusion chromatography.  Dialysis Enzyme proteins are large molecules, in size and molecular weight. This property can be used to concentrate proteins from aqueous solutions. Technically this is not a method of protein purification but a method of protein concentration. Small molecular weight solutes can be removed from proteins with the use of semi permeable membranes. One can hold mixture of proteins and other solutes in a tube or a sac made of cellophane or collidion membrane in a large body of low molarity buffer. Due to concentration gradient, water and small molecular weight solutes will move out of the sac. Normally this procedure is carried out at low temperature to protect activity of the enzyme.  ultrafiltration Another way of separating proteins from small molecules is by ultrafiltration. In this procedure, protein solution is filtered through a semi permeable membrane under pressure or using centrifugal force. Small solutes pass through membrane whereas proteins are retained on the membrane. Synthetically prepared membranes of cellophane or collidion are used for ultrafiltration. the selective permeability of a membrane structure a device or system that applies the required pressure, minimizes buildup of retained material on the filter, The surface of the UF membrane contains pores with diameters small enough to distinguish between the sizes and shapes of dissolved molecules. Those above a predetermined size range are rejected, whereas those below that range pass through the membrane with the solvent flow  Centrifugation Biologists use the technique of centrifugation to extract and isolate pure samples of individual cell organelles for study Once isolated, the chemistry and functioning of the organelles can be investigated in detail Two types of centrifugation are possible DIFFERENTIAL DENSITY GRADIENT CENTRIFUGATION CENTRIFUGATION Isolated organelles are separated Isolated organelles are separated on the basis of their weight on the basis of their density Solutions of increasing density, such The suspension of isolated organelles is as sucrose solutions, are layered into spun for different combinations of speed a test tube with the most concentrated and time solution at the bottom of the tube The lowest speed and shortest time The suspension of isolated organelles is separates out the heaviest organelles pipetted onto the top of the most dilute solution with high speeds and longer times separating the lightest organelles As the tubes are spun, the organelles collect in the layer which corresponds to their own density  Velocity centrifugation, often called rate-zonal centrifugation , separates cell components based on how fast they sediment. The sample is carefully layered on the top of the centrifuge tube. The tube is filled with a sucrose gradient, from about 5% to 30% sucrose. The sucrose forms a density gradient, which helps keep the cell components in tight bands and not diffusing. Sometimes compounds other than sucrose are used: Ficoll and Percoll are common ones. Unlike sucrose they can’t diffuse into membrane-bound organelles. The samples are then centrifuged until different bands of cell components have separated due to their different sedimentation coefficients. Note that if you keep centrifuging, eventually everything will end up in the pellet. This is NOT an equilibrium method. You can then puncture the bottom of the tube and remove the bands drop by drop.  Equilibrium centrifugation (isopycnic) separates on the basis of buoyant density: the density where the cell components float. Buoyant density is independent of size and shape. It is measured in g/ml, with water having a buoyant density of 1.0. The samples are mixed with a high concentration of sucrose or cesium chloride (CsCl), then centrifuged until everything in the mixture has floated to its equilibrium density position. The centrifugal forces generate a density gradient from the CsCl solution, because there is a large difference in the gravitational force at the top of the tube than at the bottom. This is an equilibrium technique: once the components have floated to their positions, they stay there. This is a very common technique for purifying DNA. What is the Difference Between Rate Zonal and Isopycnic Centrifugation? The key difference between rate zonal and isopycnic centrifugation is that the rate zonal centrifugation is important in separating particles that differ in size but not in their density, whereas the isopycnic centrifugation is important in separating particles that differ in density but not in their size.  Molecular exclusion chromatography (aka MSC,GFC, Gel permeation Chromatography) In this method column is prepared from inert hydrated polymeric materials such as dextran (Sephadex), agarose (Sepharose), or polyacrylamide (Bio-gel), which are available in various pore sizes as readymade materials. Column is usually prepared by washing the gel material with buffer and equilibrating with the same buffer so that beads swell and attain uniform pore size. Protein mixture is layered on the top of the column and buffer is passed over it. Proteins of different molecular sizes penetrate the beads to a different degree. And thus travel down the column at rates. Very large sized protein molecules cannot enter the pores of the beads; they are said to be excluded and thus remaining the excluded volume, defined as the volume of aqueous phase outside the beads. On the other hand very small proteins can enter the pores of the beads freely. Small proteins are retarded by the column while large proteins pass rapidly since they cannot enter the beads. Proteins of the intermediate sizes will be excluded from the beads to the degree that depends on their size. Separated proteins are collected in separate fractions and each fraction is scanned under UV light at 280 nm. ii) Based on solubility differences. This includes salt precipitation, solvent precipitation, iso-electric precipitation.  Salt precipitation: - Neutral salts such as sodium chloride, ammonium sulfate, etc. have remarkable effect on solubility of globular proteins. At low concentration, salt increases the solubility of many proteins, a phenomenon called ‘salting-in’. Salts of ammonium are far more effective at salting-in than salts of other monovalent cations, such as NaCl or KCl. In fact protein is stable in aqueous solution in presence of dilute solution of salt such as normal saline (0.85 g% NaCl).  Salting Out Most proteins are less soluble at high salt concentrations, an effect called salting out. The salt concentration at which a protein precipitates differs from one protein to another. Hence, salting out can be used to fractionate proteins. Salting out is also useful for concentrating dilute solutions of proteins, including active fractions obtained from other purification steps.  Mechanism Upon dissolution in an electrolyte solution, solvent counterions migrate towards charged surface residues on the protein, forming a rigid matrix of counterions on the protein's surface. Next to this layer is another solvation layer that is less rigid and, as one moves away from the protein surface, contains a decreasing concentration of counter-ions and an increasing concentration of co-ions. The presence of these solvation layers cause the protein to have fewer ionic interactions with other proteins and decreases the likelihood of aggregation.  Ammonium sulfate precipitation ©Wilbur H. Campbell, 1996  Repulsive electrostatic forces Repulsive electrostatic forces also form when proteins are dissolved in water. Water forms a solvation layer around the hydrophilic surface residues of a protein. Water establishes a concentration gradient around the protein, with the highest concentration at the protein surface. This water network has a damping effect on the attractive forces between proteins.  Basic residues on a protein can have electrostatic interactions with acidic residues on another protein. However, solvation by ions in an electrolytic solution or water will decrease protein– protein attractive forces. Therefore, to precipitate or induce accumulation of proteins, the hydration layer around the protein should be reduced.  Precipitate formation Occurs in a stepwise process. First, a precipitating agent is added and the solution is steadily mixed. Mixing causes the precipitant and protein to collide. Next, proteins undergo a nucleation phase, where submicroscopic sized protein aggregates, or particles, are generated. Growth of these particles.. Once the particles reach a critical size (0.1 µm to 10 µm), by diffusive addition of individual protein molecules to it, they continue to grow by colliding into each other and sticking or flocculating. During the final step, called aging in a shear field, the precipitate particles repeatedly collide and stick, then break apart, until a stable mean particle size is reached, which is dependent upon individual proteins.  Principle behind salting out of proteins 1.The concentration of any salt necessary to cause precipitation of a particular protein is related to the number and distribution of charges and of hydrophobic residues exposed and rendered dominant as the charges are neutralized. 2.This property is exploited to separate some proteins from others by precipitation at high salt concentrations. 3.As salt concentration increases, protein solubility decreases. 4.Net effect is dehydration of proteins which promotes self- association and aggregation  Pretend you are a researcher that wants to isolate a novel, unknown protein from a bacterial culture. You grow 500 ml of the bacteria overnight at 37oC and harvest the bacteria by centrifugation. You remove the culture broth and retain the bacterial pellet. You then lyse the bacteria using freeze/thaw in 10 mL of reaction buffer. You then centrifuge the lysed bacteria to remove the insoluble materials and retain the supernatant that contains the soluble proteins. Your protein of interest has a biological activity that you can measure using a simple assay that causes a color change in the reaction mixture. You also note that this reaction rate increases with increasing concentrations of your protein supernatant At this point, you can measure your baseline concentrations for the first purification level (bacterial lysis and removal of insoluble proteins and other cellular debris by centrifugation). Activity vs. Specific Activity  Mean to keep track of Enzyme purification Total Protein Total Activity ,enzyme unit, Specific Activity Yield Purification level  Precipitation with miscible solvents Addition of miscible solvents such as ethanol or methanol to a solution may cause proteins in the solution to precipitate. The solvation layer around the protein will decrease as the organic solvent progressively displaces water from the protein surface and binds it in hydration layers around the organic solvent molecules. With smaller hydration layers, the proteins can aggregate by attractive electrostatic and dipole forces. Important parameters to consider are temperature, which should be less than 0 °C to avoid denaturation, pH and protein concentration in solution. Miscible organic solvents decrease the dielectric constant of water, which in effect allows two proteins to come close together. ii) Based on solubility differences. This includes salt precipitation, solvent precipitation, iso-electric precipitation. Isoelectric precipitation The solubility of globular protein is affected by the pH of the surrounding environment.  The isoelectric point of a protein (I.E.P.) is the PH at which the molecule is electrically neutral (net charge is zero). When a protein is charged e.g. with a –ve charge, the –ve molecules repel each other and thus remain in the solution and do not precipitate. When a protein is neutral at the I.E.P. they tend to precipitate due to the absence of the repelling forces and, the high molecular weight of protein. ` Casein, like proteins, are made up of many hundreds of individual amino acids. Each may have a positive or a negative charge, depending on the pH of the [milk] system. At some pH value, all the positive charges and all the negative charges on the [casein] protein will be in balance, so that the net charge on the protein will be zero. * Principle Most proteins show minimum solubility at their isoelectric point pH of milk is 4.5-4.8 (its isoelectric point). Casein is also insoluble in ethanol and this property is used to remove unwanted fat from the preparation. Milk is present at a high PH than the isoelectric point of casein The pH at which a protein is least soluble is its isoelectric pH. Isoelectric pH is designated by the symbol ‘pI’. It can be defined as a pH at which protein has no net charge. At this pH the protein is electrophoretically neutral. Under these conditions there is no electrostatic repulsion between neighboring protein molecules and they tend to coalesce and precipitate. The pI of most proteins is in the pH range of 4–6. Mineral acids, such as hydrochloric and sulfuric acid are used as precipitants. The greatest disadvantage to isoelectric point precipitation is the irreversible denaturation caused by the mineral acids. For this reason isoelectric point precipitation is most often used to precipitate contaminant proteins, rather than the target protein. The precipitation of casein during cheese making, or during production of sodium caseinate, is an isoelectric precipitation.  Isoelectric values of some common proteins. Sr.No. Protein Isoelectric pH 1 Pepsin 1.0 2 Egg albumin 4.6 3 Serum albumin 4.9 4 Urease 5.0 5 Beta lactoglobulin 5.2 6 Gamma globulin 6.6 7 Hemoglobin 6.8 8 Myoglobin 7.0 9 Ribonuclease 9.6 10 Chymotrypsinogen 9.5 11 Cytochrome c 10.6 12 Lysozyme 11.0  Revision The isoelectric point (pI) is the pH of a solution at which the net primary charge of a protein becomes zero. At a solution pH that is above the pI the surface of the protein is predominantly negatively charged and therefore like-charged molecules will exhibit repulsive forces. Likewise, at a solution pH that is below the pI, the surface of the protein is predominantly positively charged and repulsion between proteins occurs. However, at the pI the negative and positive charges cancel, repulsive electrostatic forces are reduced and the attraction forces predominate. The attraction forces will cause aggregation and precipitation.  iii) Based on electric charge It includes ion-exchange chromatography, adsorption chromatography, electrophoresis isoelectric focusing. Ion-exchange chromatography: Proteins show acid base properties. This is shown because of their ionizable ‘R’ groups. These groups will show positive charge or negative charge depending on the pH of the surrounding solution. This feature is utilized for their separation in ion-exchange chromatography. This process was first performed by H. Sober and E. Peterson in U. S. A. in 1950s for the separation of proteins. The most commonly used materials for chromatography are synthetically derived celluloses namely DEAE cellulose and CM cellulose.  General steps for ion exchange chromatographic purification 1. Protein mixture is transferred into low ionic strength buffer (mobile phase). 2. Ion exchange adsorbent (stationary phase) is packed into a column, and the column is pre-equilibrated with the buffer of identical pH and similar ionic strength as protein mixture (preferably the same buffer as protein mixture). 3. Protein mixture is applied onto the column. Proteins charged oppositely to ion-exchange media are temporarily retained in the column. All other proteins simply pass through the column and are collected during this step. 4. Retained proteins are eluted from the column by applying a modified buffer. 5. Elution is most commonly achieved by gradually increasing ionic strength of the buffer via salt gradient, and proteins are eluted in order of increasing their net charges. 6. Is specific cases the elution can be accomplished by (a) pH change and (b) affinity methods. Adsorption chromatography In practice adsorption chromatography is very similar to ion-exchange chromatography except that adsorbent which is packed in the glass chromatography column contains both positive and negative charged groups. Adsorption v absorption  ADSORPTION CHROMATOGRAPHY is one type of process which is used for the separation of components in a mixture by introducing it into chromatography system, Based on the relative differences in adsorption of components to the stationary phase present in the chromatography column the mixture is separated.  The efficiency of the separation depends on: The solubility of the molecule in the mobile phase. Binding strength to the stationary phase. Greater the binding strength more would be the retardation of the adsorbate by the adsorbent, i.e., the molecules would move slowly. The matrix or the stationary phase can be made up of alumina or silica. Alumina can be acidic, basic or neutral whereas silica (SiO2) is acidic in nature.  iii) Based on electric charge It includes ion-exchange chromatography, adsorption chromatography, electrophoresis isoelectric focusing. The rate at which proteins move in an electrical field (migration rate, in units of cm2 V-1 sec-1) is governed by a complex relationship between the physical characteristics of both the electrophoresis system and the proteins. The strength of the electric field, the properties of the electrophoretic medium (usually a polyacrylamide gel), the temperature of the system, and the pH, ion type, and concentration of the buffer all play roles along with the size, shape, and charge of the proteins (Garfin 1990). Proteins come in a wide range of size and shapes and have charges determined by the dissociation constants of their constituent amino acids. As a result, proteins have characteristic migration rates that can be exploited for separation purposes. Protein electrophoresis can be performed in liquid or gel-based media and can also be used to move proteins from one medium to another ( blotting applications).  Protein electrophoresis can be used in a variety of applications protein purification or purity determination (for example, at various stages during a chromatographic purification), to determine size, isoelectric point (pI), and enzymatic activity, or to provide data on the regulation of protein expression. In fact, a number of different techniques can be grouped under the term protein electrophoresis Movement of proteins during including gel electrophoresis, electrophoresis. isoelectric focusing (IEF), and two-dimensional (2-D) electrophoresis. A typical protein electrophoresis workflow. About 1.4 grams of SDS bind to a gram of protein,corresponding to one SDS molecule per two amino acids. SDS acts as a surfactant, masking the proteins' intrinsic charge and conferring them very similar charge-to-mass ratios. The intrinsic charges of the proteins are negligible in comparison to the SDS loading, and the positive charges are also greatly reduced in the basic pH range of a separating gel. Upon application of a constant electric field, the protein migrate towards the anode, each with a different speed, depending on its mass. Procedure allows precise protein separation by mass.  Moving-boundary electrophoresis Moving-boundary electrophoresis (MBE also free-boundary electrophoresis) is a technique for separation of chemical compounds by electrophoresis in a free solution. The moving-boundary electrophoresis apparatus includes a U-shaped cell filled with buffer solution and electrodes immersed at its ends. The sample applied could be any mixture of charged components such as a protein mixture. On applying voltage, the compounds will migrate to the anode or cathode depending on their charges. The change in the refractive index at the boundary of the separated compounds is detected using Schlieren optics at both ends of the solution in the cell. Figure shows line drawing of Schlieren optical pattern. Each peak in the pattern corresponds to the moving boundary of a specific protein. If the electrophoretic mobility of a given protein is determined at several pH values, the isoelectric pH of the can be extrapolated.  iii) Based on electric charge It includes ion-exchange chromatography, adsorption chromatography, electrophoresis isoelectric focusing.  Based on specific binding property and selective adsorption. Affinity chromatography. Based on specific binding property and selective adsorption. Affinity chromatography.  Preparation of purification table Although, there is no much hard and fast rule for preparation of a purification table, one should show steps used for purification of the enzyme protein. In each step, amount of the total enzyme activity, total protein, specific activity, fold purification, percentage recovery are mentioned. Volume of the sample in each step can also be mentioned. A typical arbitrary purification table of starch phosphorylase is shown in Table 1.  Purification table The purpose of a purification table is to monitor the progress of the enzyme purification. Both yield and relative purity of the enzyme are calculated, taking advantage of protein concentration and enzyme activity experimentally determined for each fraction.  Enzyme Purification Table Problem A method for the purification of acid phosphatase from wheat germ is summarized in the following table. For each step, calculate specific activity, percent yield, and degree of purification (i.e. n-fold relative to the crude). Indicate which step results in the greatest purification (i.e. the largest fold).