Question

SIRJE GEL OBJECTIVE: Partial purification of a protein, isocitrate dehydrogenase (IDH), using DEAE ion exchange chromatography. IDH will be eluted from the column using increasing concentrations of KCI. IDH will be tracked using the IDH activity assay and the extent of purification will be analyzed using SDS PAGE Last week we assayed for the activity of the enzyme, isocitrate dehydrogenase, from E.coli strains carrying a plasmid that over-expresses the E. coli IDH gene. To purify proteins from cel extracts, biochemists typically use a series of protocols that successively enrich for the protein of choice. A precipitation of protein is often performed by varying pH or using high concentrations of salts (e.g. ammonium sulfate) which are then followed by one or more chromatographic approaches which include size exclusion chromatography (separates by size) and ion exchange chromatography (separates by surface charges). With ion exchange, pH extremes or high salt concentrations have to be modified to avoid interference with protein binding to the ion exchange matrix. Even without a preliminary fractionation, the sample buffer needs to be changed to a low salt buffer with the appropriate pH to allow for suitable binding of the protein to the resin; for today's experiment, this buffer will be Tris-IEC (lon Exchange Chromatography Buffer: 20 mM Tris Buffer, pH 8.5, 2 mM MgCl, 5 mM citrate, 10% glycerol). Salt removal and buffer exchange could be accomplished using desalting columns (e.g. BioRad 10DG desalting columns) or through overnight dialysis (2 or more changes of buffer). We choose to equilibrate our cell extract with Tris-IEC using dialysis which has been performed by your instructors. Refer to our supplemental reading for more on the theory and practice of ion exchange chromatography (Duong-Ly & Gabelli, 2014, Methods in Enzymology 514:95-103). EXPT 6B PROTOCOL: PART III: PREPARATION OF THE DEAE COLUMN: (WEEK 11) We will use columns containing -2 ml of a DEAE-Sepharose Fast Flow anion exchange matrix (Sigma-Aldrich Co). You will receive -2 ml of resin in Tris-IEC Buffer. With this resin, you will prepare the column as follows: 1. Gently resuspend the resin in its tube with a plastic Pasteur transfer pipet. • When resuspended, transfer all the DEAE-Sepharose resin into the column and allow liquid to flow through the column. Catch all liquid waste into a plastic beaker (waste container). Do not let the column go completely dryl Column fluid flow can be stopped at any time with a pinch clamp. Add 4 ml Tris-IEC buffer to the original resin tube. Using the transfer pipet, rinse the original resin tube and transfer the contents into the column. Allow the liquid to flow until it reaches the top of the resin. . Rinse the sides of the column (above the packed resin) with an additional Tris-IEC buffer. Allow the liquid to pass through the column until no more than 0.5-1 mm remains in the column above the matrix/resin. Do not let the column run completely dry. At any point, you can stop the flow using a pinch clamp on the bottom tubing. i. If the resin runs dry to the point of cracking, liquid loaded on top of the gel can move through the cracks and avoid entering the resin. In this event, resuspend the resin in an additional 2-3 ml of Tris-IEC buffer and allow the liquid to flow out of the column; this will repack the resin.

Answer 1

1. The pH of Tris IEC buffer here is 8.5

The pKa of DEAE is given as ~11.5 and so at a pH 8.5 which is below its pKa, DEAE would get protonated and would carry a net positive charge. DEAE is used as an anion exchange matrix where the matrix is positively charged and the counter-ions as well as proteins that interact with the matrix would be negatively charged.

2. Proteins carry a net negative charge at a pH above their pI and carry a net positive charge when subjected to a pH below their pI. Iso citrate Dehydrogenase has a pI of 5.15 and so would carry a net negative charge at pH 8.5, and would bind to the DEAE resin at this pH.