Protein Molecular Weights

In this elegant exercise, students use the technique of gel permeation chromatography to sort four substances according to their molecular weights. Low and high molecular weights are indicated by potassium ferricyanide (yellow) and blue dextran. Two proteins are compared, alpha- and beta-amylase. Since the amylases are not colored, their elution positions must be determined by assays for starch hydrolysis.

Setting up this lab each year is fairly easy, but there are several initial hurdles, mostly related to cost. All the stuff used in the exercise is costly, but once obtained, the materials should last as long as you will be in your present position. Fortunately for me, I already had quite a collection of columns and gel filtration media at hand. For columns, I use Kontes' or Bio-Rad's relatively inexpensive columns in the 1x20 cm size. For media, I use Pharmacia's Sephacryl S-300, but others like Sepharose CL-4B or Sephadex G-75 through G-200 should work.

Columns packed with a 1 x 18 cm bed of Sephacryl S-300 are stored in the refrigerator from year to year equilibrated with 33% ethanol. For use, I reequilibrate them by running a few mL of water followed by a column volume of 0.1 M NaCl. The beads need to be suspended once in the column and allowed to resettle to clear air bubbles that may form from warming to room temperature.

Students do the actual chromatography in pairs. Stock solutions of blue dextran (8 mg/mL) and potassium ferricyanide (20 mg/mL) in 0.1 M NaCl are stored from year to year in the freezer. Alpha-amylase (porcine pancreatic, Sigma #A3176) is prepared fresh (5 mg/mL in 0.1 M NaCl); it dissolves incompletely, and to avoid clogging the columns it is filtered through glass fiber filters. Beta-amylase (sweet potato, Sigma #A7005) comes as a suspension in ammonium sulfate. Sample is prepared fresh by combining one volume each of blue dextran and potassium ferricyanide with two volumes of alpha-amylase. To this mixture is added 5 microliters per mL of the beta-amylase suspension. Each pair of students is given about 0.25-0.4 mL.

At the beginning of lab, students drain their columns and then they (or I) put their sample on top without disturbing the flat top of their packed beads (necessary for best resolution). It's rinsed in carefully with 4 mL of 0.1 M NaCl; while that is draining, I go over the rest of the procedures. When the column top is again dry, they gently fill it to the top with 0.1 M NaCl and begin collecting 10 fractions of 20 drops each; this takes 45 minutes to an hour. They are asked to describe what they see as the sample moves down the column and to record in which tubes the most intense blue and yellow colors are found.

When the fractions have been collected, they must be assayed for the two amylases. One drop of each fraction is mixed in a separate tube with 6 drops of 1% starch and 1 drop of either alpha- or beta-amylase buffer. Alpha-amylase buffer is 1 M triethanolamine adjusted to pH 8.5 (Tris should work fine too); beta-amylase buffer is 1 M acetic acid adjusted to pH 4.0. These separate buffers don't absolutely discriminate between the two enzymes, but the difference is easily good enough to distinguish them. After standing for 10 minutes at room temperature, the reaction is stopped by adding 8 drops of DNSA reagent to each tube and heating in a boiling water bath for 10 minutes. Tubes are cooled and diluted by adding 4 mL of water to each and absorbance is determined using a Spectronic 20 spectrometer.

When carried out as I've described, blue dextran (M.W. 2,000,000) elutes predominantly in the second fraction, beta-amylase (M.W. 210,000) in the fourth, alpha-amylase (M.W. 54,000) in the seventh, and ferricyanide in the ninth, giving a very nice separation.

When the exercise is finished, students are asked to construct a graph showing their amylase data and indicating the fractions where the dextran and ferricyanide were most prominent. They are to identify, from their results, the colors of the ferricyanide and dextran (which I have obviously not given them any hints about, unless they find this page). They are told that the two amylases have molecular weights of 54,000 and 210,000 and asked which is which. They are also asked where they would expect thyroglobulin (Mr = 600,000) and trypsin (Mr = 25,000) to elute. They are finally given the trivial problem of how they would use the techniques in this exercise to determine whether the hemoglobin of a hypothetical anemic patient's blood formed stable tetramers.

After the experiment is over, I rinse the columns with a column volume or so of distilled water and then 33% ethanol and store them in the refrigerator.