Physical Techniques.pptx

PHYSICAL TECHNIQUES

Fractionation • Fractionation is a separation process in which certain

quantity of mixture (solid, liquid, suspension or isotope) is divided up in a number of small quantities • This composition of which varies according to the gradient • Biological molecules can be separated by this technique

• Cell fractionation is done to separate out its constituents

and different organelles present in the cell

Cell fractionation • Biochemical analysis requires disruption of the anatomy •

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of the cells Gentle fractionation techniques have been devised to separate the various cell components while preserving their individual functions Purification of cell organelles require three steps; Cell disruption Separation of different organelles using centrifugation Preparation of purified organelles

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can be disrupted by breaking open plasma membrane by various methods

• Osmotic alterations – using hypotonic solutions • Mechanical disruption by homogenization • This utilizes blender or French press

• Sonication • Disrupted cells are centrifuged to separate organelles

• If carefully applied these disruption procedures leave •

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organelles intact The suspension of cells – slurry contains a variety of membrane enclosed organelles These organelles have distinctive sizes, charge and densities Cellular organelles can now be fractionated by centrifugation Differential centrifugation, or Density gradient centrifugation

• In the early 1940s an instrument known as preparative • • • •

ultracentrifuge was developed This rotates extracts of broken cells at high speed This separates components on the basis of their sizes and densities Large units experience the largest centrifugal force and move most rapidly All these fractions are impure, contaminants can be removed by repeating centrifugation

Density gradient centrifugation

Solubility of protein • Solubility of protein in aqueous solution is a sensitive • • • •

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function of the concentration of dissolved salts The salt concentration is expressed in terms of its ionic strength, I Protein’s solubility at a given ionic strength varies with the types of ions in solution The solubility of a protein at low ionic strength generally increases with the salt concentration This phenomenon is known as salting in As the salt concentration increases, the additional counterions more effectively shield protein molecule’s multiple ionic charges and thereby increase the protein’s solubility

• However, at high ionic strengths, the solubilities of

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proteins, as well as most other substances, decrease This effect is known as salting out This is primarily the result of the competition between the added salt ions and other dissolved solutes fro molecules of solvation At high salt concentrations, so many of the added ions are solvated that the amount of bulk solvent available becomes insufficient to dissolve other solutes Hence, solute-solute interactions become stronger than solute-solvent interactions This leads to the precipitation of solute

• Salting out is the basis of one of the most commonly used

protein purification procedures • By adjusting the salt concentration in a solution containing a mixture of proteins to just below the precipitation point of protein to be purified, many unwanted proteins can be eliminated • Then, after the precipitates are removed by filtration or centrifugation, the salt concentration is increased to precipitate out the desired protein • Ammonium sulfate is most commonly used as a reagent for salting out proteins as its high solubility (3.9 M in water at 0 ˚C) permits the achievement of solutions with high ionic strengths

• Certain ions, notably I-, ClO4-, SCN-, Li+, Mg+, Ca+, and

Ba+, increases he solubilities of proteins rather than salting them out • As they tend to denature proteins • Conversely, ions that decrease the solubilities of proteins stabilize their native structures so that proteins to be salted out are not denatured

Effect of organic solvents • Water-miscible organic solvents such as acetone and ethanol, •

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are generally good protein precipitants This is because their low dielectric constants lower the solvating power of their aqueous solutions for dissolved ions such as proteins Different solubilities of proteins in these mixed solvents form the basis of useful fractionation technique This procedure is normally used near 0 ˚C or less because at high temperatures, organic solvents tend to denature proteins Some water-miscible organic solvents, however, are good protein solvents because of their high dielectric constants, e.g., DMSO (dimethyl sulfoxide) or DMF ( N, N- dimethylformamide)

Effects of pH • Proteins bear numerous ionizable groups which have a •

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variety of pK’s At a pH characteristic for each protein, the positive charges exactly balance its negative charges At this pH, proteins isoelectric point, pI, protein molecule carries no net charge In solutions of moderate salt concentrations, the solubility of a protein as function of pH is expected to be minimum at protein’s pI This phenomenon is the basis of a protein purification procedure known as isoelectric precipitation

Dialysis • One of the oldest procedures applied to the purification • •

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and characterization of biomolecules is dialysis A method used to separate dissolved molecules on the basis of their molecular size This technique involves an aqueous solution containing both macromolecules and small molecules in a porous membrane Sealing is done by knotting dialysis membrane tubing at both ends Sealed membrane is placed in a large container of lowionic-strength buffer Pores of the membrane are too small to allow diffusion of macromolecules of molecular weight greater than about 10,000

• Smaller molecules diffuse freely through the porous •

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membrane Passage of smaller molecules continues until their concentration inside the dialysis tubing and outside in the large volume of buffer are equal Continuous stirring is applied to the container Thus, the concentration of small molecules inside the membrane is reduced Equilibrium is volume dependent and is usually reached after 4 to 6 hours Concentration of small molecules can be reduced by replacing the dialysate with fresh buffer after intervals

• Dialysis membranes are available in a variety of materials • • • •

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and sizes Most common materials are collodion, cellophane, and cellulose Membrane tubing with complete molecular weight cutoffs ranging from 100 to 300,00 are available Dialysis is most commonly used to remove salts and other small molecules from solutions of macromolecules For example, proteins are often precipitated by addition of organic solvents or salts such as ammonium or sodium sulfate Since, the presence of these salts and organic solvents interfere with further purification and characterization of the molecules, they must be removed

• Dialysis is a simple, inexpensive, and effective method for •

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removing all small molecules, ionic or nonionic Dialysis is also useful for removing small ions and molecules that are weakly bound to biomolecules Protein cofactors such as NAD, FAD, and metal ions can often be dissociated by dialysis Removal of metal ions is facilitated by the addition of chelating agent (EDTA) to the dialysate A major disadvantage is that it may take several days of dialysis to attain a suitable separation

Ultrafiltration • Ultrafiltration involves the separation of molecular species •

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on the basis of size, shape, and/or charge The solution to be separated is forced through a membrane by an external force Membranes may be chosen for optimum flow rate, molecular specificity, and/or molecular weight cutoff Two obvious applications of membrane filtration are there: Desalting buffers or other solutions, and Clarification of turbid solutions by removal of micron- or submicron-sized particles

• Ultrafiltration membranes have MW cutoffs in the range of •

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100 to 1,000,000 They are usually composed of two layers: A thin (0.1-0.5 μm), surface, semipermeable membrane made from a variety of materials including cellulose acetate, nylon, and polyvinylidene, A thicker, inert, support base These filters function by retaining particles on the surfaces, not within the base matrix Filters are available with a mean pore size ranging from 0.25 to 15 μm) These filters require suction, pressure, or a centrifugal force for liquid flow

• Ultrafiltarion devices are available for macroseparaions • • • • •

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(upto 50 L) or for microseparations (milli- to microlitres) For solutions larger than a few mililiters, gas-pressurized cells or suction filter devices are used For concentration and purification of samples in the millito microliter range, disposable filters are available Some applications of ultrafiltration include: Clarification of solutions, Collection of precipitates for analysis, Harvesting of bacterial cells from fermentation broths Concentration of biomolecule solutions

• Membrane filters are divided into two categories:

• Depth filters: • Composed of paper, cotton or fiber glass • Function by trapping particles primarily in the ‘depths’ of

filter matrix • Have high load capacity of retaining particles on the surface and within the matrix • Have high flow rates, inert to most solvents and are inexpensive, but • Have ill defines and variable pore size due to random matrix and extensive adsorption

• Screen filters: • Composed of polyacrylamide and cellulose esters • Function by retaining particles on their surfaces

• Pore size ranging from 0.025 – 15 µm • Uniform pore size make them a widely used filters for

ultrafiltration • Applications: • In food industry – used to recover lactalbumin and

lactoglobulin from cheese whey, and for fruit juice clarification • In paper industry – used as a method to remove different colors and non-degradable pollutants • In textile industry – CM cellulose and polyvinyl alcohol used in textile industry are recovered by ultrafiltration

• Biotechnological uses: • For desalting buffers, enzymes or other solutions • Clarification of solutions by removal of micron sized

particles • Medical uses: • Used in hemodialysis to treat acute renal failure and drug

detoxification

Lyophilization • Also known as freeze drying • This is a drying technique that uses the process of

sublimation to change a solvent (water) in the frozen state directly to the vapor state under vacuum • The powder obtained after drying is a fluffy matrix that may be reconstituted by the addition of liquid • One of the most effective methods for drying or concentrating heat-sensitive materials • Biological solutions to be concentrated are ‘shell-frozen’ on the walls of a round bottomed flask

• Freezing is done by placing the flask (half full with

sample) in a dry ice-acetone bath and slowly rotating it as it is held at a 45˚ angle • Aqueous solution freezes in layers on the wall of the flask providing a large surface area for evaporation of water • The flask is then connected to a lyophilizer • Lyophilizer consists of a refrigeration unit and a vacuum pump • This combined unit maintains the sample at -40˚C for stability of biological materials and applies a vacuum of approximately 5-25mM • Ice formed from the aqueous solution sublimes and is pumped from the sample vial • All materials that are volatile under these conditions will be removed

• Nonvolatile materials such as proteins, buffers salts,

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nucleic acids, etc. will be concentrated into a light, sometimes fluffy precipitate Most freeze-dried biological materials are stable for long periods of time and some remain viable for years However, there are some precautions and limitations of lyophilization procedure Only aqueous solutions should be lyophilized Organic solvents should not be used as they increase the chance that the sample will melt and become denatured Organic vapors will also possibly pass through the cold trap into the vacuum pump, causing damage

Applications of freeze drying • Pharmaceutical and biotechnology: • Pharmaceutical companies often use freeze-drying to

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increase the shelf life of the products, such as vaccines and other injectables Food industry: Freeze-drying is used to preserve food products, such as freeze-dried ice cream, instant coffee and freeze dried food products used by hikers Technological industry: In chemical synthesis, products are often freeze dried to make them more stable – often reserved for materials that are heat sensitive, such as proteins, enzymes, microorganisms and blood plasma

Electrophoresis • The study of movement of charged molecules in an • • • •

electric field It can be a protein, DNA or any biomolecule It is used for characterization and analysis of charged biological molecules The movement of a molecule in an electric field is influenced by factors such as charge, mass, etc. Movement of charged molecules in an electric field is represented by an equation •

V = Eq/f

• V = velocity of charged molecules • E = electric field in (volts/cm) • q = net charge on the molecule • f = frictional fore (depends on the mass and shape of the

molecules • Under constant electric field the equation becomes • •

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• V = q/f If molecules have same shape then friction is from mass f varies with size or mass for similar conformational molecules like linear fragments of nucleic acids or spherical proteins which have globular structures Larger the size slower will be the movement Velocity depends upon charge and mass i.e., charge to mass ratio • V= q/f = e/m

Methods of electrophoresis • All types of methods are based on the same principle •

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discussed earlier The major difference between the methods depends upon the type of support medium It can be either cellulose or thin gels Cellulose is used as a support medium for low-molecularweight biochemicals such as amino acids and carbohydrates Whereas, gels are used as support medium for larger molecules These gels are made up of polyacrylamide or agarose

Polyacrylamide gel electrophoresis (PAGE) • Polyacrylamide gels are made by the polymerization if two • •

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compounds Acrylamide, and bisacrylamide Gels are prepared by the free radical polymerization of acrylamide and the cross-linking agent N,-N’-methylenebis-acrylamide Chemical polymerization is controlled by an initiatorcatalyst system, ammonium persulfate-N,N,N,N’tetramethylethylenediamine (TEMED) Polymerized gels have several positive features in electrophoresis

1. High resolving power for small and moderately sized

proteins and nucleic acids 2. Acceptance of relatively large sample sizes 3. Minimal interactions of the migrating molecules with the matrix 4. Physical stability of the matrix • Resolving power of a gel depends upon the concentration of acrylamide and bisacrylamide in the gel • Lower concentrations – larger pores, hence allowing analysis of higher-molecular-weight biomolecules • Higher concentrations – smaller pores, allowing analysis of lower-molecular-weight biomolecules • If larger pore size gels are present then molecules will move easily (less hindered) and will not be resolved correctly

• Polyacrylamide electrophoresis can be done using either

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of the two arrangements: column or slab Slab gels are now more widely used than column gels Several samples can be analyzed on a slab gel rather than using several column gels The polyacrylamide slab is prepared between two glass plates – separated by spacers Spacers allow uniform slab thickness A plastic comb is inserted into the top of the slab gel during polymerization to form wells Gel electrophoresis is usually carried out at basic pH, at which most biological polymers are anionic hence they move towards the anode

• A “tracking dye” is also applied, which moves more rapidly

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through the gel than the sample components When the dye band has moved to the opposite end of the gel, voltage is turned off, gel is removed and stained with a dye A protein marker which is a mixture of known molecular weight molecules is used for the recognition of proteins Proteins are usually separated on polyacrylamide gel while nucleic acids are separated on agarose gels but both can either be used The hazardous effect of polyacrylamide gel is that the monomer acrylamide is a neurotoxin and a cancer suspect agent Several manufacturers now offer gels precast in glass or plastic cassettes

Discontinuous gel electrophoresis • This is also known as “disc electrophoresis”

• It is a modification of PAGE • Three significant characteristics of this method are that; • There are two gel layers, a lower or resolving gel and an

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upper or stacking gel, The buffers used to prepare the two gel layers are of different ionic strengths and pH, The stacking gel has lower acrylamide concentration so its pore size is larger These changes cause the formation of highly concentrated bands of sample in the stacking gel, And a greater resolution of the sample components in the lower gel

• Sample concentration in the upper gel occurs in the • • • • •

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following manner The sample is usually dissolved in glycine-chloride buffer, pH 8 to 9 before loading Glycine at this pH exists in two forms, a zwitterion and an anion Average charge on glycine anions at pH 8.5 is about -0.2 When the sample reaches the stacking gel which has a pH of about 6.9, glycine shifts towards zwitterionic form Since most of the protein and nucleic acid samples are still anionic at this pH, they replace glycinate as mobile ion Relative ion mobilities in the stacking gel therefore becomes, chloride > protein/ nucleic acid > glycinate

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sample will tend to accumulate and form a thin, concentrated band sandwiched between the chloride and glycinate ions • Since acrylamide concentration is low (2-3%) in stacking gel, there is little impediment on larger sample molecules • When ionic front reaches lower gel (pH 8-9), anionic glycinate and chloride ions carry most of the current • Sample molecules now encounter both increase in pH and decrease in pore size • Relative rate of movement of anions now become chloride > glycinate > protein • Each component of sample has a unique charge/mass ratio and discrete size and shape, which directly influence its mobility

Sodium dodecyl sulfate-Polyacrylamide gel (SDS-PAGE) • If the protein samples are treated so that they have a

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uniform charge, electrophoretic mobility then depends primarily on size The molecular weights of proteins can be estimated if they are subjected to electrophoresis in the presence of a detergent Sodium dodecyl sulfate (SDS) is used for this purpose It binds to proteins and disrupts the secondary, tertiary and quaternary structure to produce linear polypeptide chains Mercaptoethanol, a reducing agent reduces disulfide bonds – denaturing electrophoresis

• SDS imparts a negative charge on all molecules and

masks the protein’s intrinsic charge • So that SDS treated proteins tend to have an identical charge-to-mass ratio and similar shapes • The relative mobilities of proteins on such gel vary linearly with the logarithm of their molecular masses • Larger molecules are retarded by the molecular sieving effect of the gel and smaller molecules have greater mobility • In practice, a protein mixture is treated with 1% SDS and 0.1M mercaptoethanol in electrophoresis buffer • SDS-PAGE is valuable for estimating the molecular weight of protein subunits

Agarose Gel Electrophoresis • Nucleic acids up to 350,000 daltons (500 bp) in molecular •

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size cannot be separated by small pore size gels The standard method used to characterize RNA and DNA in the range 200 to 50,000 base pairs (50 kilobases) is electrophoresis with agarose as the support medium Agarose, a product from seaweed, is a linear polymer of galactopyranose derivatives Gels are prepared by dissolving agarose in electrophoresis buffer Nucleic acids can be visualized on gels after separation by soaking in a solution of ethidium bromide

• The mobility of agarose gel is influenced

by the agarose concentration and the molecular concentration and molecular conformation of nucleic acids • Like proteins, nucleic acids migrate at a rate that is inversely proportional to the logarithm of their molecular weights • Hence, molecular weights can be estimated from electrophoresis results using standard nucleic acids or DNA fragments of known molecular weight • The rapid advances in our understanding of nucleic acid structure and function in recent years is due primarily to the development of agarose gel electrophoresis as an analytical tool