In the Laboratory
edited by
the microscale laboratory
Arden P. Zipp SUNY-Cortland Cortland, NY 13045
Rapid Microscale Isolation and Purification of Yeast Alcohol Dehydrogenase Using Cibacron Blue Affinity Chromatography Chad Morgan and Neil Moir Chemistry Department, California State Polytechnic University, San Luis Obispo, CA 93406 A procedure is presented for the rapid and microscale isolation of yeast alcohol dehydrogenase (ADH). It provides a fast (2-h), easy, and inexpensive method for the isolation of an enzyme, as the affinity gel may be recycled, yeast extracts can be easily prepared using inexpensive equipment, and all of the steps can be performed at room temperature. The procedure lends itself well to the undergraduate biochemistry laboratory experience because it emphasizes many classical biochemical techniques such as affinity chromatography, spectrophotometry, polyacrylamide gel electrophoresis, and linked assays. Yeast ADH catalyzes the NAD+ -dependent oxidation of ethanol to acetaldehyde and NADH. The strong absorbance of NADH at 340 nm (measured with a Spectronic 20) is used to measure the activity of the enzyme, the change in specific activity that occurs during purification, and kinetics of the purified enzyme. An earlier paper in this Journal described the kinetics of this enzyme. CH3CH2OH + NAD +
CH3CHO + NADH + H + (1)
Experimental Procedure Our initial preparations involve large-scale yeast cell lysis using a Bead Beater and 0.5-mm glass beads purchased from Biospec Products (P.O. Box 722, Bartlesville, OK 74005). A half gram of fresh yeast cake is mixed together with 1 gram of 0.5-mm glass beads and 1 mL of isolation buffer (pH 6.5, 0.02 molar tris-HCl buffer containing 5 mM MgCl2, 0.4 mM EDTA, 5 mM 2mercaptoethanol) in a 2-mL microfuge tube. The mixture is alternately vortexed and chilled in an ice bath for a total of 10 min. The resulting yeast extract is microfuged at full speed (6000 × g) for 5 min to sediment the unbroken cells, denatured protein, glass beads, and other insoluble components. The supernatant may be used immediately or stored for up to a month at –20 °C with only minimal loss in activity. Before the next step, a small amount of sample (50 µL) is saved for enzyme and protein assays. Interfering proteins are precipitated with polyethylene glycol (PEG-8000). A 40% (w/w) PEG solution is added to crude supernatant in a microfuge tube to a final concentration of 8%. The tube is then capped and gently inverted 10 times to thoroughly mix the two solutions. After 5 min at room temperature, the suspension is centrifuged at full speed for 2 to 3 minutes. Eight percent PEG precipitates many high molecular weight proteins, thereby increasing the amount of yeast extract that can be applied to the affinity dye column. Additionally, the PEG supernatant can be applied directly to the column without a desalting step, thus sav-
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ing time. A Cibacron Blue F 3GA column with a 1-mL bed volume (5-mL disposable pipette fitted with a very small glass wool plug) is activated before the experiment by passing 5 column bed volumes of 6 M urea through it, followed by equilibration with 5–8 volumes of isolation buffer. One half to one milliter of the 8% PEG supernatant is carefully added to the column and allowed to fully enter the column bed. Unbound proteins are eluted from the column with 5.0 mL of isolation buffer. This fraction should contain little or no alcohol dehydrogenase. ADH is eluted by adding 2.5 mL of elution buffer (isolation buffer containing 5 mM NAD+); the first 0.5mL fraction is discarded [Esterday and Esterday (2)]. YADH elutes from the column in the next two 1-mL fractions. Generally the major part of the enzyme elutes in the first 1-mL fraction (fraction #2). Typical purification data are listed in Table 1. To measure enzymatic activity, samples were diluted in isolation buffer containing 0.5% bovine serum albumin. Fractions were diluted in 0.9% NaCl for protein determinations.1 To follow the purification of alcohol dehydrogenase visually, each fraction can be electrophoresed in a 12% polyacrylamide gel under native conditions (5). YADH is located in all fractions using a linked assay procedure. The gel is placed into a small volume of assay solution containing 0.1 mg/mL oxidized phenazine methosulfate (PMS) and 1 mg/mL Nitroblue tetrazolium sodium (NBT). After a brief development period, YADH is located by the presence of blue bands corresponding to the reduced blue formazan dye. CH3CH2OH
NAD +
PMSox
NBT
CH3CHO
YADH NADH
PMSred
Blue ppt.
Table 1. Purification of Alcohol Dehydrogenase from a Vortex Glass Bead Cytolysate of 0.5 g Bakers Yeast Cakea Fraction
Volume (µL)
Enz Act (IU)
Protein (mg)
Crude
1000
260
40
6.5
1.0
8% PEG sup
1150
255
21
12.1
1.9
Fraction 2
1000
178
1.0
Specific Act.
178
Purification
27.5
a The enzyme assay mixture used was 0.1 M ethanol, 0.01 M NAD+ , 0.1 mg/mL BSA in a 0.05 M tris buffer pH 8.5 (3 ). Protein concentration was determined using the Bradford method and corrected for yeast protein (4).
Journal of Chemical Education • Vol. 73 No. 11 November 1996
In the Laboratory
Subsequent silver staining of the same gels was used to detect contaminating proteins present in each fraction (6). After silver staining, we found the crude and 8% PEG supernatant lanes to contain 10 to 20 intense (and many minor) bands. The Cibacron Blue column– purified fraction usually contains just one intense band and a few minor ones. This electrophoretic procedure dramatically demonstrates the purification of alcohol dehydrogenase from contaminating proteins. The reader may wish to consult reference texts tor details and tips on the protein purification techniques described in this article. Two such texts are listed below (4, 7). Acknowledgment Cibacron Blue F3G-A was donated by Calzyme, Inc.
Note 1. Samples were diluted in 0.9% NaCl to yield final concentrations of 50–500 µg of protein per milliliter. One hundred microliters of the diluted sample was added to 900 µL of Bradford’s reagent (60 mg Coomassie brilliant blue dissolved in 100 mL of 2% perchloric acid). Absorbance was measured after 3 min at 610 nm.
Literature Cited 1. Utecht, R. E. J. Chem. Educ. 1994, 71, 436–437. 2. Esterday, R. L.; Esterday, J. M. Adv. Exp. Med. Biol. 1974, 42, 123–133. 3. Scopes, R. K.; Griffiths-Smith, K.; Millar, D.G. Anal. Biochem. 1981, 118, 284– 285. 4. Scopes, R. K. In Protein Purification: Principles and Practice, 3rd ed.; Cantor, C. R., Ed.; Springer Verlag: New York, 1993; p 350. 5. Hames, D. B.; Richwood, D. Gel Electrophoresis of Proteins: A Practical Approach, 2 ed.; IRL: New York, 1990; p 35. 6. Heukeshoven, J.; Dernick, R. Electrophoresis 1985, 6, 103–112. 7. Harris, E. L. V.; Angal, S. Protein Purification Methods: A Practical Approach; IRL: New York, 1989.
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