Biochemistry 462a Proteins: Purification and Characterization Reading - Chapters 5 and 6 Practice problems - Chapter 5: 8,12-14,18; Proteins extra

a problems

Protein Purification

Source of Protein o In order to purify a protein you need a source. o It might be blood or some other biological fluid, but most often it is a cell, usually a specific type - liver, muscle, yeast, bacteria, etc. o The cells must be broken open - homogenized - to release the protein in a soluble form. o Homogenization conditions must be worked out that release the protein from the cell without damaging the protein. o Membrane-bound proteins can also be purified, but different approaches are required.

Fractional Precipitation o In concentrated salt solutions, usually ammonium sulfate is used; some proteins are more soluble than others. By varying the concentration of ammonium sulfate, one can achieve some limited purification of proteins. This technique is often used in the first step of protein purification. In general, o Small proteins are more soluble than large proteins. o The larger the number of charged side chains, the more soluble the protein. Column Chromatography o The invention of column chromatography was a critical event in biochemistry, because it was the basis for the development of procedures for obtaining pure proteins. o Studies on pure proteins are essential for understanding the structural and functional properties of proteins. o In column chromatography an adsorbent (see below) is placed in a glass tube. o A protein mixture is passed into the column and binds to the adsorbent. o By proper choice of the eluting buffer, specific proteins can be eluted from the adsorbent and separated from other proteins in the mixture. o By repeating this procedure with several different adsorbents, pure protein can be obtained. o o Because proteins are not very stable, low temperature (4 C) and neutral pH must often be employed. o The properties of some adsorbents are described below.

Ion Exchange Chromatography o Ion exchange resins have fixed charges - either positive or negative. o Proteins bind to the resin via electrostatic interactions. o The strength of these interactions depends on the net charge on the protein, which is a function of pH and the nature of the weak acid amino acid side chains, and the salt concentration of the buffer - high salt concentrations reduce the interaction. Affinity Chromatography o This is a more specific interaction in which a ligand specifically recognized by the protein of interest is attached to the column material. o When a mixture of proteins is passed through the column, only those few that bind strongly to the ligand will stick, while the others will pass through the column. o By changing the buffer one weakens the interaction between the protein and the ligand, which causes the protein to be eluted from the column. o A variation is immunoaffinity chromatography; in which an antibody specific for a protein is immobilized on the column and used to affinity purify the specific protein. Gel Filtration Chromatography (= "molecular sieve" or "size exclusion chromatography") o The column consists of material that separates proteins based on their size and shape. o A wide range of molecular exclusion limits is available for separating proteins of all sizes. o For any particular column dimensions and material, the volume of buffer required to elute a specific protein depends on the molecular weight of the protein. Thus, one can separate proteins by size. o If one calibrates the column by determining the elution volume of proteins with known molecular weights, then a calibration curve relating elution volume and molecular weight can be constructed.

Such a calibration curve can then be used to estimate the molecular weight of an unknown protein.

Dialysis/Ultrafiltration o Semipermeable membranes are available, which allow passage of small molecules but exclude the passage of proteins. Sacs made of such material allow the salt and buffer components of a protein solution to be changed to another buffer

Practical Example

This figure illustrates several of the techniques discussed above. It is taken from "Isolation, Characterization, and cDNA Sequence of Two Fatty Acid-Binding Proteins from the Midgut of Manduca sexta Larvae". A. F. Smith, K. Tsuchida, E. Hanneman, T. C., Suzuki, and M. A. Wells, J. Biol. Chem. 267, 380-384 (1992). This is the elution profile from an anion exchange resin (binds negatively charged proteins). The proteins were eluted by increasing the NaCl concentration in the eluting buffer. Total protein was measured by determining the absorbance at 280 nm. In order to follow the fatty acid-binding proteins, they were labeled by binding radioactive fatty acids (CPM=counts per minute - gray shading). The purity of each peak was assessed using SDS-PAGE (insert). There are two, nearly pure, proteins that bind fatty acids. The two proteins were obtained in pure form following one additional step.

Protein Characterization

Electrophoresis o In an electric field a protein or other charged macromolecule will move with a velocity that depends directly on the charge on the macromolecule and inversely on its size and shape. Gel electrophoresis is carried out in some supporting media, usually polyacrylamide or agarose, which has pores of sufficient size to allow passage of the macromolecule. o The proteins in the gel are easily stained for detection purposes. o Because the net charge on a protein and its molecular weight are characteristic properties of a protein, electrophoresis is a powerful method for characterizing the purity of a protein preparation. SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) is a variant of electrophoresis in which the buffers contain SDS, a detergent that binds to proteins. o Most proteins bind SDS at a constant ratio, about 1 SDS for every 2 amino acids. o The large negative charge resulting from the bound SDS masks the native charge on the protein, so that all proteins have essentially the same charge to mass ratio. o This means that that the rate of movement in the electric field depends only on the polypeptide chain molecular weight. Mercaptoethanol is used to reduce disulfides. o In addition, the SDS causes all proteins to adopt a random-coil structure, which means that shape does not affect movement through the gel. o Thus SDS-PAGE is very useful method for determining the molecular weight of the individual polypeptide chains of a protein. Western blotting is a technique for detecting a specific protein in a mixture. Gel electrophoresis and SDS-PAGE electrophoresis are primarily useful as analytical techniques, although they can be used for purification. o In this figure, lane 1 would contain standards of known molecular weight, lane 2 a mixture of three unknown proteins and lanes 3-5 the three unknown proteins. o SDS-PAGE can be used to determine the molecular weight of a protein. o The molecular weights of the three unknown proteins can be determined from a calibration curve constructed by plotting the log of the molecular weight of the standard proteins vs. the distance traveled in the gel. o The distance traveled depends on the porosity of the gel. The lower the porosity the faster (further) the proteins move.

Depending on the molecular of the unknown proteins, different porosity gels will need to be used.

Isoelectric Focusing o In this technique electrophoresis occurs through a stable pH gradient. o Under these conditions the proteins will migrate in the electric field until they reach a point in the pH gradient where their net charge becomes 0 - the isoelectric point (pI). o The isoelectric point depends on the exact number and type of weak acid amino acid side chains present in the protein. Therefore, isoelectric focusing is a useful purification procedure. o Often isoelectric focusing is combined with SDS PAGE in two-dimensional electrophoresis. Molecular Weight and Shape are fundamental physical properties of a protein. o Estimates of molecular weight can be obtained using SDS-PAGE or gel filtration, as described above. o One very useful technique for measuring molecular weight and shape is centrifugation. ! A particle that is subjected to a centrifugal field by being spun in a centrifuge is subjected to a force, , where m is the mass of the mass of the particle, r is the distance of the particle from the center of rotation, and is the angular velocity. is the buoyancy factor which accounts for the fact that particle is buoyed up by the surrounding solvent of density (g/ml). is the specific volume of the particle (ml/g) (= 1/density of the particle). ! ! If = then the particle will not move. The movement of the particle through the solvent is resisted by a frictional coefficient, f, that depends on the shape of the particle. ! The frictional coefficient is an important factor in any transport process, such as centrifugation or gel filtration. ! A spherical particle has a f = 1.0, whereas a cigar-shaped or cylindricallyshaped particle will have f > 1.0. The movement of any particle under the influence of a centrifugal field is characterized by its sedimentation coefficient, S, which is directly proportional to its molecular mass, M, and inversely proportional to f. , where N is Avogadro's number.
!

Ultracentrifugation is used in two ways to characterize proteins In sedimentation equilibrium experiments, the centrifuge is operated at a relative low speed so that the forces of sedimentation and diffusion balance and the protein distributes in the centrifuge cell in a manner proportional to its molecular weight. In sedimentation velocity experiments, the centrifuge is operated at maximal speed, which causes the protein to sediment to the bottom of the tube. The rate at which the boundary moves gives S, which when combined with M gives f, a measure of the shape of the protein.

Three Dimensional Structure

Whenever possible, it is highly desirable to obtain the three dimensional structure of a protein. Most often, this is done by X-ray crystallography, although NMR can be used, especially with small proteins (< about 25,000 daltons). It is impressive to note that more than 10,000 structures have been determined, most in the last decade, as new, more powerful instruments have become available.

Structural Homology

In addition to sequence homology for proteins with identical functions from different organisms, there are often domains in a protein that are conserved. For example, most proteins that bind nucleotides, such as ADP, have a common nucleotide-binding motif.

There are even a few cases in which proteins with entirely different functions have very similar three-dimensional structures, as shown below for lysozyme, an enzyme, and lactalbumin, a milk protein.

Lysozyme

Lactalbumin