In the Laboratory
Determination of the Subunit Molecular Mass and Composition of Alcohol Dehydrogenase by SDS-PAGE
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Barbara T. Nash Division of Natural Sciences and Veterinary Technology, Mercy College, Dobbs Ferry, NY 10522;
[email protected] Polyacrylamide gel electrophoresis (PAGE) is a versatile biochemical technique that can be used to monitor specific proteins during purification, cloning (1), or changes in gene expression (2). It can be used to determine the subunit molecular mass (1, 3), subunit composition and stoichiometry (3, 4), and the isoelectric point (3, 5) of proteins. Gel electrophoresis can be used as a preparative technique to purify native proteins (6) or to isolate individual subunits (6, 7). In combination with immunoprecipitation and autoradiography, it has been used to investigate the synthesis, sorting, and processing of proteins (7, 8). In tandem with Western blotting, it is useful for identifying specific proteins in complex mixtures (9). SDS-PAGE is a simple, rapid technique that is readily adaptable to the undergraduate laboratory. Applications that have been used in undergraduate biochemistry laboratory curricula include assessment of protein purity (10), determination of subunit molecular mass (11, 12), determination of subunit stoichiometry (13), investigation of the presence of intermolecular disulfide bonds (14), and Western blotting (15–17). However, some care is required to set up an experiment to obtain accurate results and to teach students skills that they can use in a professional setting. To estimate molecular mass accurately, one must choose a gel and standards that are appropriate for the molecular mass range of the proteins being analyzed. To obtain sharp bands, sample overloading must be avoided. Standards and unknown proteins should be treated in the same manner and interpolation should be done within a linear region of the standard curve (1, 18, 19). Although it is possible to investigate the presence of intermolecular disulfide bonds by performing SDSPAGE in the presence and the absence of a reducing agent (14, 20), great care must be taken in performing this experiment (1). In the absence of a reducing agent, artefactual multimers can appear on the gel if the protein and SDS concentrations are not optimized. Moreover, if intramolecular disulfide bonds are present, proteins will not form the standard rodlike shape and their electrophoretic mobilities will be affected. This article describes the use of SDS-PAGE to determine the subunit molecular mass and composition of yeast alcohol dehydrogenase (ADH) employing state-of-the-art methods that produce accurate results. Two types of standard proteins are used. Precision Plus Protein standards consist of recombinant proteins of precisely known molecular masses that produce a ladder of very sharp bands on SDS gels. In contrast to fixed percent gels that produce standard curves that are linear only across a narrow molecular mass range, the standard curves generated by gradient gels are linear across the entire range of the standards. Kaleidoscope Prestained standards, which are prepared by the covalent attachment of dye molecules to the protein, are used solely to monitor separation during electrophoresis. Owing to heterogeneity within 1508
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each sample, these standards produce bands that are very broad and are not suitable for accurate molecular mass determinations (1, 19). Dithiothreitol (DTT) is used as the reducing agent because it is less malodorous than β-mercaptoethanol. It is used in the same concentration as that present in the standards. Precise quantities of protein are applied to the gel to determine the optimal quantity of protein required to obtain accurate measurements. A standard curve is produced using Microsoft Excel software. Students calculate the true subunit molecular mass of the protein using several pieces of information from the ExPASy protein knowledgebase (21). They use published data from gel filtration chromatography (22) to determine the molecular mass of the native protein and use this information and their data to determine the subunit stoichiometry of the protein. The subunit composition of the yeast V-ATPase complex has been investigated using SDS-PAGE and Western blotting (23). The focus of that experiment was less quantitative than the experiment reported here. Prestained molecular mass standards were used both to monitor protein transfer and to construct the standard curve. The broad bands obtained do not give accurate molecular mass determinations. Subunit stoichiometry could not be determined by this method because some of the subunits present in the complex were not detected. Materials Trizma base, glycine, SDS, glycerol, DTT, and bromophenol blue may be obtained from Sigma Chemical Company or from Bio-Rad Laboratories. Sodium phosphate and yeast ADH were obtained from Sigma. Premixed running buffer, Laemmli sample buffer, Kaleidoscope Prestained standards, Precision Plus Protein standards, Bio-Safe stain, and 4–15% Tris-HCl gradient gels were purchased from Bio-Rad. Mini-gel electrophoresis units and power supplies can be purchased from Bio-Rad, or from other vendors. Systems for airdrying gels are available from Bio-Rad or Invitrogen. Procedure Students prepare SDS-complexes of ADH in gel sample buffer. While the sample is boiling, they prepare the gel and assemble the electrophoresis unit as demonstrated by the instructor. As soon as the run begins the students will see the progressive stacking of the Kaleidoscope standards. The instructor can explain this process and role of the lead ion (chloride) and the trailing ion (glycine) in the system while the students watch this occur. When the dye front reaches the region of the gel containing more restrictive pores, the Kaleidoscope standards will begin to resolve. The instructor can then explain the process of destacking. While the gel is staining (30 minutes), the instructor can discuss how to collect
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In the Laboratory
and analyze the data. Students are instructed to select for their measurements the lane containing ADH with the sharpest band that is clearly visible and the lane containing Precision Plus Protein standards that is closest to this lane. The standard curve can be prepared using semilog paper or with a graphing software program. The gel can be documented either by photographing it with a digital camera or by scanning the dried gel. The digital file can be incorporated into the students’ lab reports. Hazards The running buffer contains Tris, glycine, and SDS, which may cause skin and eye irritation. Laemmli sample buffer contains Tris, glycerol, and bromophenol blue, which may cause skin and eye irritation. Glycerol, SDS, and bromophenol blue are harmful if swallowed, absorbed through the skin, or inhaled. The gel sample buffer contains DTT, which is neurotoxic and harmful if swallowed. It is irritating to the eyes, respiratory system and skin and has an unpleasant odor. Concentrated solutions should be handled in a fume hood and gloves should be worn. Yeast ADH may cause skin and eye irritation and may be harmful if inhaled. It may be irritating to mucous membranes and the upper respiratory tract and it may be harmful if swallowed. Prolonged or repeated exposure may cause allergic reactions in certain sensitive individuals.
Figure 1. Photograph of a student’s gel: lanes 2 and 9–Kaleidoscope standards; lanes 5 and 8–Precision Plus Protein standards; and lanes 3, 4, 6, and 7–0.25, 0.50, 1.0, or 2.0 µg of ADH, respectively. Note that the angle at which the photograph was taken has distorted the appearance of lane 2.
Results and Discussion A photographic image of a student’s gel is shown in Figure 1. An image obtained from scanning the dried gel is very similar (Figure 2). The Kaleidoscope standards (lanes 2 and 9) produce very broad bands, whereas the Precision Plus Protein standards (lanes 5 and 8) produce sharp bands. Because ADH (lanes 3, 4, 6, 7) appears as a single band, it can be concluded that it is either a monomeric protein or a homologous multimer. The bands in lanes 4, 6, and 7, containing 0.50 µg, 1.0 µg, and 2.0 µg, respectively, are broad. Students conclude that because all of the protein runs in a single location, these lanes are overloaded. The band in lane 3, containing 0.25 µg, is sharp and distinct and contains the optimal quantity of protein for obtaining accurate measurements. The standard curve obtained from the migration distances of the standards in lane 5 (Figure 3) has an r 2 value of 1.00. The subunit molecular mass of ADH determined from the average of 15 similar student experiments was 38.6 ± 1.3 kDa (4% error). The true subunit molecular mass is 36,996 Da (21). The molecular mass of the native protein, calculated from data obtained from chromatography on Sephacryl S200 (22), is 150 kDa. This value agrees with that obtained from equilibrium centrifugation (24). The number of subunits present in the native protein is calculated by dividing the molecular mass of the native protein by the experimental value of the subunit molecular mass and rounding to the nearest whole number. From this, it can be concluded that ADH is a homologous tetramer, a fact that is confirmed on the ExPASy Web site. Although it would be preferable for students to obtain the molecular mass of the native protein experimentally, by gel filtration chromatography or by perwww.JCE.DivCHED.org
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Figure 2. Image of the gel shown in Figure 1, obtained by scanning the dried gel. The spot over lane 2 is an air bubble that was trapped between the cellophane sheets when the gel was dried.
Figure 3. Standard curve for the subunit molecular mass determination of ADH by SDS-PAGE. Data was obtained from the protein standards in lane 5 of the gel shown in Figure 1. Molecular masses of the standards are 250, 150, 100, 75, 50, 37, 25, 20, 15, and 10 kDa.
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In the Laboratory
forming SDS-PAGE on the cross-linked and uncross-linked samples of ADH (13), this would require more lab time. Moreover, gel filtration chromatography of ADH would require facilities that are frequently not available in undergraduate institutions (a cold room and a class set of fraction collectors, peristaltic pumps, and UV spectrophotometers).
Table 1. Results of Student Sur vey Statement
Mean
σ
• I understood the main concepts of the lab.
4.6
0.8
• This lab improved my understanding of material covered in the lecture.
4.5
0.8
• I understood why we were doing each activity.
4.5
0.8
Student Evaluation
• My understanding of the methods of biochemistry has improved.
4.5
0.6
An anonymous survey was conducted during the fall semester of 2005 to evaluate students’ opinion of this lab. A 100% response was obtained from 35 students who performed the lab. The students were asked to indicate the degree to which they agreed or disagreed with the statements shown in Table 1 by selecting one of the following ratings: strongly agree (5), agree (4), neither agree nor disagree (3), disagree (2), strongly disagree (1). Clearly students learned from and valued the lab experience.
• I have a better understanding of the role of computer technology in biochemistry.
4.6
0.6
• My ability to use computer technology in the biochemical lab has improved.
4.3
0.8
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• My biochemical laboratory skills have improved.
4.6
0.6
• I feel confident that I could repeat this experiment successfully.
4.7
0.8
• I feel capable of troubleshooting problems that might arise with this technique.
4.2
1.0
NOTE: Scale for the mean is 1–5, with 1 strongly disagree and 5 strongly agree.
Supplemental Materials
Student handouts, notes for the instructor, and answers to a postlab problem set are available in this issue of JCE Online. Acknowledgments Funding to purchase the computers and software used in this experiment came from a National Science Foundation grant (DUE-0088496, Nancy L. Beverly, PI; Barbara Nash, Co-PI). The author wishes to thank the students in the Mercy College biochemistry lab during the fall semesters of 2003, 2004, and 2005 for the use of their data and their helpful feedback on the lab instructions. I would also like to thank P. V. Minorsky (Mercy College) for helpful comments regarding the preparation of the manuscript and Natalie B. Bronstein (Mercy College) for her expert assistance in preparing some of the graphics files. Literature Cited 1. Shi, Q.; Jackowski, G. One-Dimensional Polyacrylamide Gel Electrophoresis. In Gel Electrophoresis of Proteins: A Practical Approach, 3rd ed.; Hames, B. D., Ed.; Oxford University Press: New York, 1998. 2. Inamine, G.; Nash, B.; Weissbach, H.; Brot, N. Proc. Natl. Acad. Sci. U.S.A. 1985, 82, 5690–5694. 3. Tate, S. S.; Meister, A. Proc. Natl. Acad. Sci. U.S.A. 1976, 73, 2599–2603. 4. Davies, G. E.; Stark, G. R. Proc. Natl. Acad. Sci. U.S.A. 1970, 66, 651–656. 5. Righetti, P. G.; Bossi, A.; Gelfi, C. Conventional Isoelectric Focusing in Gel Slabs, in Capillaries, and Immobilized pH Gradients. In Gel Electrophoresis of Proteins: A Practical Approach, 3rd ed.; Hames, B. D., Ed.; Oxford University Press: New York, 1998. 6. Lee, K. H.; Harrington, M. G. Preparative Gel Electrophoresis. In Gel Electrophoresis of Proteins: A Practical Approach, 3rd ed.; Hames, B. D., Ed.; Oxford University Press: New York, 1998. 7. Nash, B.; Tate, S. S. J. Biol. Chem. 1984, 259, 678–685.
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8. Blobel, G.; Dobberstein, B. J. Cell Biol. 1975, 67, 835–851. 9. Hanash, S. M. Two-Dimensional Gel Electrophoresis. In Gel Electrophoresis of Proteins: A Practical Approach, 3rd ed.; Hames, B. D., Ed.; Oxford University Press: New York, 1998. 10. Roberts, C. A.; Jones, C.; Spencer, E. J.; Bowman, G. C.; Blackman, D. J. Chem. Educ. 1976, 53, 62–63. 11. Choinski, J. S., Jr. Experimental Cell & Molecular Biology, 2nd ed.; Wm. C. Brown: Dubuque, IA, 1992; pp 155–164. 12. Farrell, S. O.; Ranallo, R. T. Experiments in Biochemistry: A Hands-on Approach; Thomson Learning, Inc.: Pacific Grove, CA, 2000; pp 229–242. 13. Moe, O. A., Jr.; Smith, G. J. Chem. Educ. 1977, 54, 392–393. 14. Powers, J. L.; Andrews, C. S.; St. Antoine, C. C.; Jain, S. S.; Bevilacqua, V. L. H. J. Chem. Educ. 2005, 82, 93–95. 15. Farrell, S. O.; Farrell, L. E. J. Chem. Educ. 1995, 72, 740–742. 16. Boyer, R. Modern Experimental Biochemistry, 3rd ed.; Benjamin/Cummings: San Francisco, 2000; pp 321–331. 17. Parra-Belky, K. J. Chem. Educ. 2002, 79, 1348–1350. 18. Molecular Weight Determination by SDS-PAGE, tech note 3133; Bio-Rad Laboratories: Hercules, CA, 2004. 19. Using Precision Plus Protein Standards To Determine Molecular Weight, tech note 3144; Bio-Rad Laboratories: Hercules, CA, 2004. 20. van Holde, K. E.; Johnson, W. C.; Ho, P. S. Principles of Physical Biochemistry, 2nd ed.; Pearson Education: Upper Saddle River, NJ, 2006; pp 261–262. 21. ExPASy Protein knowledgebase. http://www.expasy.ch/sprot/ (accessed Jun 2007). 22. Gel Filtration Molecular Weight Markers, Technical Bulletin No. GF-3; Sigma Chemical Co.: St. Louis, MO, Oct 1987; p 7. 23. Parra-Belky, K.; McCulloch, K.; Wick, N.; Shircliff, R.; Croft, N.; Margalef, K.; Brown, J.; Crabill, T.; Jankord, R.; Waldo, E. Biochem. Mol. Biol. Educ. 2005, 33, 289–292. 24. Kägi, J. H. R.; Vallee, B. B. J. Biol. Chem. 1960, 235, 3188– 3192.
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