1 SEPARATION AND PURIFICATION OF PROTEINS AND ENZYMESAKINBO D.B. Lecture Series

2 Proteins Proteins are large, nitrogenous organic compounds with complex molecules composed of one or more long chains of amino acids that play many critical roles in the body. They occur especially as structural components of body tissues such and as enzymes and antibodies and they do most of the work in cells and are required for the structure, function, and regulation of the body's tissues and organs.

3 Protein Separation The separation of proteins involves exploiting differences in the solubility of the particles in aqueous solutions. Protein molecule solubility is determined by its amino acid sequence because this determines its size, shape, hydrophobicity and electrical charge. Antibodies are powerful reagents used to detect, quantify, and isolate proteins and are used in affinity chromatography combined with gel electrophoresis in Western blotting, a powerful method for separating and detecting a protein in a mixture.

4 Protein Separation.. Proteins can be selectively precipitated or solubilized by altering the pH, ionic strength, dielectric constant or temperature of a solution. These separation techniques are the most simple to use when large quantities of sample are involved as they are relatively quick, inexpensive and are not particularly influenced by other macromolecules or components.

5 PROTEIN PURIFICATION The purification of protein involves a series of processes employed in isolating one or a few proteins from a complex mixture such as cells, tissues or whole organisms. It is an essential technique by which a single protein type is isolated from a complex mixture such as a cell lysate and is vital for the characterization of the function, structure and interactions of the protein of interest. Protein Purification is the process of separating proteins for individual analysis and is the second step of studying proteins sequel to the identification and separation. Prior to purification, it is essential that protein be released from cell by homogenization and many steps/techniques are expedient to this extraction and separation of protein of interest from many contaminants. Separation techniques – size, charge and polarity.

6 Purification of proteins

7 Purification of proteinsThe table above shows different purification procedures involved in the purification of enzyme Xanthine dehydrogenase showing the recovery and purity of protein obtained with each procedure. The specific activity column compares the purity of protein at each step and percentage recovery shows how much protein has been retained at each step. The retention of protein drops at each step but the purity increases.

8 Protein purification For purification to occur, the protein must be gotten into solution; if the protein is located in the cytosol, the cell would need to be opened. For animal cells, this can be accomplished with osmotic lysis. The cells will be placed in hypotonic solution; less solutes than inside the cell. While cells with a cell wall (bacteria, plants) require the use of other methods such as the use of lysozyme for bacteria as it is often effective by selectively degrading bacterial cell wall. Detergents or organic solvents can also be used, although these are capable of denaturing the protein.

9 Procedure for protein purificationChoose source of proteins. Solubilize proteins. Stabilize proteins. Specific assay for protein of interest: enzymatic activity, immunological activity, physical characteristics (e.g. molecular mass, spectroscopic properties, etc.), biological activity. Assay should be specific, rapid, sensitive, and quantitative.

10 Mechanical processes of cell breakage.High speed blender Homogenizer: i. Sonication – Use of sound waves to break open cells. ii. Continuous freezing and thawing – Ruptures cells French press Sonicator Grinding tissue in a blender with a suitable buffer releases soluble proteins and various subcellular organelles. Potter-Elvejhem homogenizer – A thick walled test tube with a tight fitting plunger breaks open cells – organelles intact. After the cells have been broken, the crude lysate, may be filtered or centrifuged to remove the particulate cell debris. The protein of interest is in the supernatant.

11 Protein purification For the purification of proteins that are components of membranes or subcellular assembly, it is essential to remove the assembly from the rest of the cellular material (mitochondria for example). This is carried out by differential centrifugation-cell lysate is centrifuged at a speed that removes only the cell components denser than the desired organelle followed by a centrifugation at speed that spins down the organelle.

12 Protein Assays Protein assays are carried out all through the purification process to ensure that the protein of interest is present. In a situation where the protein of interest is an enzyme, using a reaction for which that enzyme is a catalyst is a good way to monitor protein recovery. In order to monitor the increase of the product of the enzymatic reaction, the following can be employed; i. Fluorescence ii. Production of acid to be monitored by titration iii. Coupled enzymatic reaction - couple with another enzyme to make an observable color change or substance.

13 Protein Assays.. The use of immunochemical techniques to assay for proteins involves the use of specific immunoglobulins (antibodies), proteins that interact specifically with the protein of interest and can be easily monitored. Antibodies are produced by an animal’s immune system in response to the introduction of a foreign protein called antigens. Antibodies specifically bind to the foreign protein and are extracted from blood serum of animal that has been immunized against a particular protein. Many different antibodies in sera with different specificities and binding affinities toward the protein of interest.

14 Protein purification

15 Protein solubility The solubility of proteins is affected by multiple acid-base groups on protein and therefore dependent on concentrations of dissolved salts, the polarity of solvent, the pH and the temperature. Certain proteins are precipitated from solutions under conditions which others remain soluble-so we can use this as an initial purification step of proteins. Salting out or salting in procedures take advantage of ionic strength. Ionic strength (I) = 1/2ciZi Ci = molar concentration of ionic species Zi = ionic charge

16 Protein solubility.. The solubility of proteins at a given ionic strength varies with the types of ions in solution and the order of effectiveness of these various ions in influencing protein solubility is quite similar for different proteins and is due to the ion’s size and hydration. Protein solubility at low ionic strength generally increases with the salt concentration and this process is called salting-in. As the salt concentration increases, the additional counterions more effectively shield the protein molecule’s multiple ionic charges and thereby increasing the protein’s solubility.

17 Protein solubility.. At high ionic strengths, proteins and most other substances decrease their solubilities and this is called salting-out which results from a competition between the added salt ions and the other dissolved solutes for molecules of solvation.

18 Protein precipitation using Ammonium Sulfate

19 Protein solubility.. From the above table, at around 30% ammonium sulfate about 80% of the total protein concentration in our sample can be precipitated, yet the activity assay for the desired protein indicates that about 95% of this protein is still soluble. Therefore, at 80% ammonium sulfate all of our desired protein has been precipitated. From these results, it is instructive to: 1. Add ammonium sulfate to our sample to a concentration of 30% saturation 2. Centrifuge and discard the pellet 3. Add ammonium sulfate to 80% saturation 4. Centrifuge and keep the pellet. Re-suspend the pellet in buffer to solubilize the protein. A 5-fold purification with about 95% yield is thus expected with this method.

20 Protein solubility. Salting-out is one of the most commonly used protein purification procedures and this is achieved by adjusting the salt concentration in a solution with a mixture of proteins to just below the precipitation point of the protein to be purified, many unwanted proteins can be eliminated from solution. Upon the removal of the precipitate by filtration or centrifugation, the salt concentration of the remaining solution is increased to precipitate the desired protein. Ammonium sulfate is the most commonly used reagent, having a high solubility (3.9 M in water at 0 ºC) and a high ionic strength solution can be made (up to 23.5 in water at 0 ºC). However, some certain ions such as I-, Li+, Mg2+, Ca2+ and Ba+ increase the solubility of proteins rather than salting out and are capable of denaturing proteins too.

21 Protein solubility.. Water-miscible organic solvents also precipitate proteins such as Acetone and ethanol. Low dielectric constants lower than the solvating power of their aqueous solutions for dissolved ions thereby increasing precipitation. This technique is done at low temperatures (0 ºC) because at higher temperatures, the solvent evaporates and can magnify the differences in salting out procedures.

22 Protein solubility.. Proteins have various ionizable groups. At a pH characteristic for each protein, the positive charges on the molecule exactly balances the negative charges and this is called the isoelectric point; pI. At pI, the protein has no net charge and is immobile in an electric field hence influencing the solubility by changes in the pH.

23 Solubility of b-lactoglobin as a function of pH at several NaCl concentrations. The solubility of the lactoglobin protein is worst when the pI of beta lactoglobin is at a pH of We see here that in diluted NaCl solutions the solubility changes based on the pH.

24 Protein solubility.. A protein in a pH near its isolectric point is not subject to salting in because as the pH is moved away from the pI of the protein, the protein’s net charge is increased and it becomes easier to salt in. Salts inhibit interactions between neighboring molecules in the protein that promote aggregation and precipitation. pI’s of proteins can be used to precipitate proteins.

25 Isoelectric points of some common proteins.

26 Column chromatographyThe mixture of proteins to be fractionated is dissolved in a liquid or gaseous fluid called the mobile phase and this solution is passed through a column consisting of a porous solid matrix called the stationary phase. These are sometimes called resins when used in liquid chromatography. This stationary phase would usually have certain physical and chemical characteristics that allow it to interact in various ways with different proteins. The common types of chromatographic stationary phases are: Ion exchange Hydrophobic Gel filtration Affinity

27 Ion exchange chromatographyIon exchange resins contain charged groups. If these groups are acidic in nature they interact with positively charged proteins and are called cation exchangers. If these groups are basic in nature, they interact with negatively charged molecules and are called anion exchangers. Positively charged (basic) protein or enzyme + + CH2-COO- + CH2-COO- + CM cellulose cation exchanger - Negatively charged (acidic) protein or enzyme CH2-CH2 -NH+(CH2CH2) CH2-CH2 -NH+(CH2CH2) DEAE cellulose anion exchanger

28 Ion exchange chromatographyFor protein binding, the pH is fixed (usually near neutral) under low salt conditions. Example cation exchange column… Positively charged protein or enzyme bind to the column + CM cellulose cation exchanger CH2-COO- + + + - - Negatively charged proteins pass through the column

29 Ion exchange chromatographyTo elute our protein of interest, add increasingly higher amount of salt (increase the ionic strength). Na+ will interact with the cation resin and Cl- will interact with our positively charged protein to elute off the column. + CM cellulose cation exchanger CH2-COO- + Increasing [NaCl] of the elution buffer Cl- CM cellulose cation exchanger CH2-COO- Na+ Na+2 + Cl- Na+ Cl- Na+2 Cl-

30 Ion exchange chromatographyProteins will bind to an ion exchanger with different affinities and as the column is washed with buffer, proteins with relatively low affinities for the ion exchange resin will move through the column faster than the proteins that bind to the column. The greater the binding affinity of a protein for the ion exchange column, the more it will be slowed in eluting off the column. Proteins can be eluted by changing the elution buffer to one with a higher salt concentration and/or a different pH (stepwise elution or gradient elution). Cation exchangers bind to proteins with positive charges. Anion exchangers bind to proteins with negative charges.

31 Ion exchange chromatography using stepwise elution.

32 Ion exchange chromatography using stepwise elution.From the above diagram, the tan region of the column represents the ion exchanger and colored bands indicate the various proteins. a). The protein mixture is bound to the topmost portion of the ion exchanger in the chromatography column. b). As the elution progresses, the various proteins separate into discrete bands as a consequence of their different affinities for the ion exchanger under the prevailing solution conditions. Here the first band of protein (red) has passed through the column and is being isolated as a separate fraction, whereas the other, less mobile, bands remain near the top of the column. c) The salt concentration in the elution buffer is increased to enhance the mobility of the proteins and thus elute their remaining bands. d) The elution diagram of the protein mixture.

33 Ion exchange chromatographyGradient elution can improve the washing of ion exchange columns. The salt concentration and/or pH is continuously varied as the column is eluted so as to release sequentially the proteins bound to the column. The most widely used gradient is the linear gradient where the concentration of eluent solution varies linearly with the volume of the solution passed. The solute concentration, c, is expressed as: c = c2 - (c2 - c1)f c1 = the initial concentration of the solution in the mixing chamber c2 = the concentration of the reservoir chamber f = the remaining fraction of the combined volumes of the solutions initially present in both reservoirs.

34 Device for generating a linear concentration gradient.c = c2 - (c2 - c1)f

35 Some Biochemically Useful Ion Exchangers.

36 Ion exchange chromatographyIon exchangers can be cellulosic ion exchangers and gel-type ion exchangers. Cellulosic ion exchangers are most common. Gel-type ion exchangers can combine with gel filtration properties and have higher capacity. Disadvantage of these materials is that they are easily compressed so eluent flow is low. There are other materials derived from silica or coated glass beads that address this problem.

37 Gel filtration chromatographyThis is also called size exclusion chromatography or molecular sieve chromatography. Assuming proteins are spherical, this process will work thus; The gel bead has a varied molecular size holes such that small molecules like water and buffer can enter them completely. size Molecular mass (daltons) 10,000 30,000 100,000

38 Gel filtration chromatographySome proteins are small enough to also enter the molecular holes of the gel bead while other proteins are too large to enter the holes and bye-pass the gel bead. This concept is of Reverse Sieve, since a normal sieve retains large and passes small particles. In gel filtration the larger proteins elute first, medium sized ones next and finally the smallest proteins elute last.

39 Gel filtration chromatographyflow

40 Gel filtration chromatographyflow In gel filtration the larger proteins elute first.

41 Gel filtration chromatographyflow

42 Gel filtration chromatographyflow Medium sized proteins elute next.

43 Gel filtration chromatographyflow Finally, the smallest elutes last.

44 Gel filtration chromatographyThe molecular mass of the smallest molecule unable to penetrate the pores of the gel is at the exclusion limit. The exclusion limit is a function of molecular shape, since elongated molecules are less likely to penetrate a gel pore than other shapes. Behavior of the molecule on the gel can be quantitatively characterized. Total bed volume of the column Vt = Vx + V0 Vx = volume occupied by gel beads V0 = volume of solvent space surrounding gel; Typically 35%

45 Gel filtration chromatography..Elution volume (Ve) is the volume of a solvent required to elute a given solute from the column after it has first contacted the gel. Relative elution volume (Ve/V0) is the behavior of a particular solute on a given gel that is independent of the size of the column. This effectually means that molecules with molecular masses ranging below the exclusion limit of a gel will elute from a gel in the order of their molecular masses with the largest eluting first.

46 Gel filtration chromatography.

47 Some Commonly Used Gel Filtration Materials

48 Affinity chromatographyMany proteins can bind specific molecules very tightly but noncovalently and this is advantageous with affinity chromatography. Glucose (small dark blue molecule) binding to hexokinase. The enzyme acts like a jaw and clamps down on the substrate (glucose)

49 Affinity chromatographyFunctionality of the Affinity chromatography Ligand - a molecule that specifically targets and binds to the protein of interest. Inert support + + Spacer arms Ligand Inert support Affinity material prepared

50 Affinity chromatographyInert support Mixture of proteins Inert support Unwanted proteins

51 Affinity chromatographyInert support Elute with competitive ligand. Inert support Remove from competitive ligand by dialysis.

52 Affinity chromatographyTo remove the protein of interest from the column; Elute with a solution of a compound with higher affinity than the ligand (competitive) Change the pH, ionic strength and/or temperature so that the protein-ligand complex is no longer stable.

53 Immunoaffinity chromatographyMonoclonal antibodies can be attached to the column material. The column only binds the protein against which the antibody has been raised. 10,000-fold purification in a single step! Disadvantages Difficult to produce monoclonal antibodies. Harsh conditions to elute the bound protein