Gel-Filtration Chromatography of Proteins
This exercise is a variation on an exercise from my other biochemistry course, CHM 2201. Students use gel-filtration chromatography to separate three proteins whose molecular weights differ significantly and whose activities are simple to measure, namely, alpha- and beta-amylase and lysozyme. The enzymes separate cleanly into three peaks even under the low-resolution conditions they use in the lab. They can carry out the separation and assays all in one period if they don't mess around.
I set up in advance 1 x 17 cm columns of Sephacryl S-300 in economy columns available from either Bio-Rad or Kontes. The columns are equilibrated with 0.1 M NaCl. The sample mixture (which I prepare) contains lysozyme (crystalline; 5 mg/mL sample), pancreatic amylase (alpha-amylase, Sigma cat. # A3176; 5 mg/mL sample filtered through glass fiber filter), and beta-amylase (Sigma cat. # A7005; suspension in ammonium sulfate, 4 microliters/mL sample).
Students put 0.4 mL of sample on top of the column; I caution them about the importance of not disturbing the surface. They wash the sample onto the column with 4 mL of 0.1 M NaCl, discarding the eluate. Then they elute the column with 0.1 M NaCl, collecting ten fractions of twenty drops each.
Lysozyme assay. Students use the lysozyme assay described elsewhere.
Amylase assays. These assays differ only in the buffer used. Students put six drops (0.30 mL) of 1% starch and one drop of buffer in a test tube and start the reaction by adding a drop of sample. After ten minutes at room temperature they stop the reaction by adding eight drops (0.40 mL) of DNSA reagent. Color is developed by placing tubes in a boiling water bath for ten minutes. Then the sample is diluted with 4.0 mL of water and absorbances are determined at 540 nm.
The buffer for alpha-amylase is pH 8.5; I use 1 M triethanolamine adjusted with HCl, but Tris would probably work fine. The buffer for beta-amylase is 1 M acetate at pH 4.
Students are instructed to plot all their data on a single graph and to estimate the position of peaks to "tenths of a fraction" by eyeball. I then give them the known molecular weights of lysozyme (14,400) and beta-amylase (210,000) and ask them to determine the molecular weight of alpha-amylase (54,000) by plotting the log of molecular weight versus fraction number. Some came out remarkably close.
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