Protein Separation Techniques William R. Dayton* Introduction same tissue or cell often yield information about how the proteins interact in vivo. In food systems, the interaction Protein separation techniques are based on the widely between specific proteins can affect their functionality in the different chemical, physical and biological properties of var-- system. Consequently, it is often of great interest to ascertain ious protein molecules. The number and complexity of these whether two purified proteins interact and how this interac- techniques are rapidly increasing as our understanding of tion affects their properties. protein molecules expands. Currently, many specialized col- umn materials for use in chromatographic separation of Protein Purification Techniques proteins are commercially available. Additihally, sophisticat- ed fraction collection and protein detection equipment is A. Gel Permeation Chromatography (1-4) being used to facilitate protein separation. The purpose of Gel permeation chromatography separates protein mole- this review is to briefly outline some of the common methods cules according to their size and shape, with large, currently being used for separating proteins. It should be nonspherical molecules eluting more rapidly than small, emphasized that the large and rapidly growing technology spherical molecules. Gel permeation resins may be thought associated with protein separation cannot be adequately of as beads containing numerous uniform conical pits on covered in this brief review. Consequently, it is intended to their surface. Molecules that are larger than the diameter of provide a base of information from which interested readers the pit do not enter it and consequently pass through the can expand their studies of specific protein separation tech- column quickly. Small molecules can enter the pits. The niques. smaller the molecule, the deeper it can go into these conical pits and the longer it will remain there. Consequently. the Experimental Uses for presence of the pits impedes the progress of small molecules Protein Separation Techniques through the column more than it does the progress of large molecules. Protein separation techniques have traditionally been In order to standardize a gel permeation column, it is used to isolate and to purify specific proteins in order to necessary to calculate the fraction of the stationary gel facilitate studies of their enzymatic, physical, chemical and volume that is available for diffusion of a given solute species structural properties. These kinds of studies are necessary in (KaJ This is done by using the following parameters: order to elucidate the biological role of individual proteins in elution volume (VJ -the volume at which a the cell and to understand the mechanism by which the given protein elutes activity of specific enzymes are controlled. total volume (V,) -volume of the packed Because protein separation techniques are based on the column bed chemical, physical and enzymatic properties of proteins, the void volume (V,) -elution volume of the behavior of a specific protein during a separation protocol molecules only can reveal a great deal about that protein. For example, ion distributed in the mobile exchange chromatography can give an indication of the phase of the gel because relative net charge on the protein at a given pH; gel perme- they are larger than the ation chromatography can be used to determine molecular diameter of the largest size (Stokes radius): affinity chromatography can be used to pits in the gel. analyze the interaction of a protein with specific substrates, ve - vo inhibitors, activators or antibodies; and hydrophobic interac- K,” tion chromatography can be used to examine the hydropho- v, - vo bicity of specific proteins. Kavfor a given protein is proportional to the log of the Protein purification techniques can also be used to moni- molecular radius of that protein. Under optimal conditions, tor the interactions of specific purified proteins. Studies of in protein molecules do not bind to gel permeation resins. vitro interactions between two or more proteins from the Both column length and flow rate affect the degree of resolution obtainable using gel permeation chromatography. Since resolution is directly proportional to the square root of ‘W R. Dayton, Andrew Boss Laboratory, 7354 Eckles the column length. a relatively longer column is desirable. Avenue, University of Minnesota, St. Paul, MN 55108 Resolution is also improved by reducing the flow rate of the Reciprocal Meat Conference Proceedings. Volume 36, column. Because increasing column length and decreasing 1983. flow rate both increase the time required to elute the protein 98 36th Reciprocal Meat Conference 99 of interest from a gel permeation column, this increased as elution proceeds and more eluent is added to the time factor must be balanced against the increased column. A given protein is released from a particular portion resolution it provides. of the column when the pH in that region of the column reaches the isoelectric pH of the protein. The protein then B. lon-exchange Chromatography (5-1 1) moves through the column as additional exchanger is An ion exchanger consists of an insoluble support matrix titrated to the isoelectric pH of the protein. Elution of containing chemically bound positive (anion exchanger) or different proteins is in descending order of their isoelectric negative (cation exchanger) groups and mobile counter points. ions. The counter ions may be reversibly exchanged with other ions of the same charge without altering the insoluble matrix or the chemically bound groups. C. Hydrophobic Interaction Chromatography (12). Protein molecules bind electrostatically and reversibly to In this type of chromatography, proteins are separated ion exchangers if their net charge is opposite to that of the based on the strengths of their hydrophobic interactions with exchanger. All proteins are amphoteric polyelectrolytes, an uncharged resin containing hydrophobic groups. These which means that their net charge is dependent upon the interactions are facilitated by increasing ionic strength. Con- pH of the solution in which they are suspended. At low pH, sequently, proteins are often adsorbed to a hydrophobic resin the net charge on most proteins is positive (binds to cation in the presence of high concentrations of neutral salt (e.g., exchangers) and at high pH the net charge is usually NaCI). negative (binds to anion exchangers). At the point of zero Selective elution of adsorbed proteins is achieved by net charge, the isoelectric point, protein molecules are not altering the eluent in a way that causes desorption based on bound to any type of ion exchanger. Since protein the strength of the hydrophobic interaction of individual pro- molecules can bind to either anion or to cation exchangers teins with the hydrophobic matrix. This can be accomplished (depending upon pH), the stability of the protein at various by lowering the ionic strength of the eluent, lowering the pH’s is usually the most important factor in choosing an ion polarity of the eluent by including substances such as ethyl- exchanger for specific protein separations. ene glycol, including detergent in the eluent, or raising the pH Once proteins have been bound to an ion exchange of the eluant. resin, selective elution can be accomplished in several ways. For example, altering the column buffer pH towards the isoelectric point of a specific protein will cause that D. Affinity Chromatography (13-14) protein to lose its net charge, desorb and elute from the Affinity chromatography is a type of adsorption chroma- column. Consequently, if a pH gradient is applied to an ion tography in which the column bed material has biological exchanger containing bound protein, each protein will elute affinity for the particular protein to be isolated. Specific as its isoelectric point is reached and protein separation will adsorptive properties of the bed material are obtained by be accomplished. Changes in ionic strength of the column covalently coupling an appropriate binding ligand to an in- buffer can also be used to selectively release proteins from soluble matrix. The covalently attached ligand then binds a ion exchange resins. At low ionic strength, competition for specific protein from a complex mixture of proteins and the charged groups on the ion exchanger is at a minimum and bound protein is retained on the matrix. Unbound proteins even slightly charged proteins are bound strongly. are then washed away and the bound protein is released by Increasing the ionic strength of the column buffer (by using altering the composition of the eluent. Highly selective pro- a linear gradient) increases the competition and interferes tein isolations are achieved due to natural biological specific- with the interaction between the ion exchanger and the ities. For example. enzymes can be purified by using affinity protein. Weakly charged molecules (those closest to their columns containing a substrate analog, specific inhibitor or isoelectric pH) are eluted at lower ionic strength than more cofactor for that enzyme. Antigens can be purified by using highly charged molecules. affinity columns containing the appropriate antibody. Similar- Chromatofocusing is a specialized form of ion-exchange ly, antibodies can be purified on affinity columns containing chromatography that requires a resin containing charged covalently bound antigen. groups with high buffering capacity. The column is Affinity