The central theme of the biochemical approach is to rebuild functional systems from purified components. This allows for the greatest confidence in assigning functional roles to cellular components. The drawback, however, is that you must purify those components before you can experiment.

While the particular methods will vary depending on what you are purifying, the central tennant always remains the same—discrimination. At the most fundamental level, every purification method discriminates between molecules based on some physical property. In biochemical purifications the list of physical properties may include, molecular weight, net charge, binding affinity, resistance to heat, or differential solubility. Often two or more of these properties are exploited in series to effect a full purification of the molecule of interest from a crude homogenate.

While there are many physical properites which can be used for separation, we will focus today on ion exchange. Ion exchange is a chromatography method in which the stationary phase is an inert bead (to make it stationary) derivatized with an ionizable functional group. While many commercial variations are available, there are just two functional types: anion and cation exchange resin. Anion exchange resin is positively charged and will hold (and exchange) anions. Cation exchange resin is negatively charged and will attract cations. Thus, the resins are named for the ions that bind to them, not their intrinsic charge.

The "exchange" in ion exchange refers to the method used to release absorbed ions from the resin. Typically the sample is loaded onto the column in a very low concentration of salt. This allows the ions in solution to associate with the ion exchange resin. To release the bound ions, the ionic strength of the buffer is increased. As the salt (often sodium chloride, but not necessarily) concentration increases, bound ions are now in competition for binding with smaller, higher charge density ions. Eventually (at high enough salt concentrations) the biological ions will lose the competition to the inorganic ions and be released from the column and collected in a purified form. The exact salt concentration required to release any particular biological ion is unique, providing for separation among similarly charged ions.

Ion exchange was used, in part, in the purification of pneumococcal surface protein a as reported in Xia H, Yu J, Sun Q, Luc J, Gu T, Guo X, Li B, Chen X, Zhang K, Konga W and Wu Y (2018) Expression and purification of pneumococcal surface protein a of clade 4 in Escherichia coli using hydroxylapatite and ion-exchange column chromatography Protein Expres Purif 151 56-61.

Today we will be demonstrating anion exchange chromatography. The anion exchange resin is diethylaminoethyl (DEAE) sephadex. Sephadex is the inert bead and each bead is derivatized with multiple DEAE groups. The DEAE residue is a tertiary amine which will bear a formal positive charge at most pH values below pH 10. An illustration of DEAE resin is shown below.

While purifying enzymes would be the most biochemical, it isn't going to be the most illustrative, primarly because few proteins absorb light in the visible spectrum. In order to aid with the visualization of the process, we will absorb and release the dye bromophenol blue, providing an easily visualized bright blue. Bromophenol blue has a the structure illustrated below, the negatively charged sulfate group in the upper right (when the sodium cation dissociates in solution).

The first task is to pour the column. Remove the lid from the column. Pick up tube of resin and gently suspend the settled resin into a thin slurry. Pour all the resin into the column and replace the lid on the column.

While the resin will settle in the column, given enough time, it will pack faster under flow. Fill the upper resevoir with Tris (loading) buffer and open the stopcock at the bottom of the column. As buffer begins to drip out of the column, buffer should begin to flow up the tubing and, eventually, begin dripping into the column. The tube may not fill completely, or have air bubbles. That is fine, as long as the whole system is flowing. If there is no flow, or it looks like there is air entering the system, check your connections and try again. It is best to have some buffer on top of the resin bed in the column, you may safely stop the flow and add buffer above the column, if necessary. Allow the column to pack and equilibrate until the upper resevoir is just empty (you can safely chase the last drops with the tube).

To load the column, fill the upper resevoir with your bromophenol blue solution and allow it to run into the column. As it drips in, note the pattern of retention in the column.

When you are finished loading the column, you'll note that, while the resevoir is empty, there is still a sizable volume of dye in the tube and above the resin. To address this, the column, once loaded, is washed with a large volume of loading buffer in order to make sure the system only contains loading buffer and material absorbed to the resin. Wash your column by filling the resevoir with loading buffer and allowing it to run through your column. Notice that bromophenol blue remains largely on the column as you wash. Continue washing the column with another full resevoir of loading buffer.

With a washed column, it is time for elution. Elution is the process in which smaller, more charge dense ions exchange with our "biological" molecules absorbed to the column. To exchange chloride anions for bromophenol blue anions, fill the resevoir with buffer + 2.0 M NaCl and allow it to run through the column.

You can watch the buffer on top of your column for the lines of mixing to know when the salt has hit the column. Shortly thereafter, as you watch the dye being released, switch collection to a clean vessel to collect bromophenol blue from the column.

1. Based on visual inspection in lab, would the column have a greater binding capacity than what we utilized? That is, do you think the column would have been able to absorb more bromophenol blue? Explain.
2. A unique feature of proteins is that they are a polymer made of monomer units which, in their variable regions, may be negatively charged, electrostatically neutral or positively charged. As a result, both the sign and the magnitude of the net charge depends on the sequence of the protein and the pH in question. For example, bovine pancreatic ribonuclease (RNase A) has a positive net charge at pH 7, but a negative net charge at pH 9. Would you use an anion or cation exchange residue to attract RNase A at pH 7?
3. Elution in the experiments was by a direct increase in salt concentration. It is also possible to gradually increase the salt concentration to produce a linear increase in salt concentration. This will allow the release of some proteins at very low salt concentrations followed by other proteins at higher salt concentrations. What might differ about these proteins that some would exchange at low and others at higher salt concentrations, all other things (pH, buffer, temperature) being equal?

Provide answers to the questions above.