This exercise serves as an introduction to the ideas behind protein purification and determination of enzyme specific activity. Students prepare lysozyme from egg white using ion exchange chromatography. The procedure is simple and requires little time or complicated equipment. It takes advantage of lysozyme's remarkably high pI (over 11); even well above neutral pH, lysozyme has a net positive charge, resulting in its retention by a cation exchange matrix when other proteins in egg white would run through due to a net negative charge. Cation exchange matrices that might be used include phosphocellulose and carboxymethyl- or sulfopropyl-substituted cellulose, Sephadex, or Sepharose (I use S-Sepharose). For cation exchange chromatography, the buffer used should consist of neutral and anionic species. You could go high-tech and use a Good buffer such as TAPS, CHES, or CAPS, but I recommend glycine, which is cheap and has a pKa of around 9.5, which is ideal for this application.

I prepare the egg white mixture ahead of lab by diluting an egg white with five volumes of 25 mM glycine, pH 9.2; even at only 25 mM, the buffer holds the pH at 9.2. I shear the mixture and homogenize gently with a teflon-glass homogenizer, strain through four layers of cheesecloth, and finally, just before the lab period, filter through a fast-flow paper (such as Fisher P8 or Whatman #4). The concern is that if the albumin coagulates, which it does quite readily, it will clog the chromatography column.

Students load 5 mL of this filtered mixture onto a 3-mL bed of S-Sepharose equilibrated with the glycine buffer and wash it on with 4 mL of glycine buffer. They then elute the column with 4 mL portions of glycine buffer containing 0.10 M and 0.75 M NaCl. Incidentally, the flow rate can be increased substantially, without otherwise interfering with the separation, by hanging a 3-4 inch length of plastic tubing (such as PE-205 attached to a blunted 16-gauge hypodermic needle) to the bottom of the column. Lysozyme elutes in the 0.75 M NaCl fraction. The egg white mixture and their various eluates are analyzed for lysozyme and protein as described below.

Lysozyme assay: The substrate is a suspension of 0.20 mg/mL of Micrococcus lysodeikticus in 0.1 M potassium phosphate (pH 7.5). The absorbance at 450 nm should be between 0.6 and 0.8. The bacteria are available commercially as a lyophilized powder that is infinitely stable stored in the freezer. Students determine the absorbance (450 nm) of 3 mL of substrate, then add 0.10 mL of sample, invert quickly to mix, and record the absorbance at precisely 30 seconds after addition. I don't attempt to control temperature. If you have a recording spectrophotometer, you can actually measure the tangent at zero time, but the above approach works adequately to get a rate.

Protein assay: I use the biuret assay. Students prepare a standard curve using 0, 2, 4, and 6 mg/mL gelatin. Using 0.1 M NaCl, I dilute the original egg white mixture 5-fold and the students dilute their first run-through with an equal volume of 0.1 M NaCl before carrying out the protein assay. Students should check their results to be sure the values stay within the standard curve; if not, have them dilute samples appropriately.

For their write-up, I give the students a definition of a unit of lysozyme activity as the amount of enzyme that gives a decrease of 1.0 absorbance unit in one minute. They then have to complete a table for the original egg white and all their eluates listing (1) total volume, (2) protein concentration, (3) total protein, (4) lysozyme concentration (units/mL), (5) total lysozyme units, and (6, for original egg white and eluate with highest activity only) lysozyme specific activity (units/mg). Finally, they are to calculate recoveries of protein and lysozyme and the increase in specific activity between egg white and the eluate with highest activity.

After the exercise, I wash the columns with a couple column volumes of 0.1 M NaOH, then glycine buffer containing 0.2 mg/mL of sodium azide for storage.

Revised 6/11/07