In the Laboratory
Utilizing Isolation, Purification, and Characterization of Enzymes as Project-Oriented Labs for Undergraduate Biochemistry S. Todd Deal and Michael O. Hurst Department of Chemistry, Georgia Southern University, Statesboro, GA 30460 The development of an undergraduate into a chemist is a difficult process, and chemistry programs foster it in a variety of ways. In general, students are expected to develop steadily as they move through individual courses and through the curriculum as a whole. This development is usually designed to focus on growth both in chemical knowledge and in the maturity necessary to function independently. For example, in the organic chemistry laboratory, students move from learning about techniques such as recrystallization and distillation, to intricate syntheses, and finally to working independently to solve unknown structures. Also, in most chemistry programs, as students move through the curriculum they go from simply recording data in freshman lab reports, to keeping professional notebooks in organic chemistry, to preparing publication-type lab reports in analytical and physical chemistry. Biochemistry does not play a major role in most traditional chemistry programs because of its status as an elective in the chemistry major. At Georgia Southern, we are attempting to increase the role of biochemistry in the professional development of our students by restructuring the course and redesigning the laboratory experience. Our first effort to increase the “visibility” of the biochemistry program was to expand it from one quarter to a full year program, with two quarters of lab. By making this change, we felt that we could more thoroughly present the details that make biochemistry fascinating. In addition, we would be able to better demonstrate the interdisciplinary links between the chemical and biological sciences. As this change became a huge success (evidenced by the rapid growth in the enrollment in the courses) we began to implement the second part of our plan—the updating of our laboratory experiences. A quick survey of the past five years of this Journal turned up many new and innovative laboratory experiences for biochemistry (1–9). In fact, one article outlined a complete modern laboratory program designed to teach undergraduates the techniques of modern biochemistry (10). We found all of these labs to be carefully thought out and well-designed; however, we felt that they were lacking in one area we wished to emphasize in our program—student growth through independence in the laboratory setting. After months of brainstorming with our departmental colleagues and careful consideration of the abilities of our students, we developed a program that we believe helps students develop understanding of and familiarity with modern biochemical techniques. We took special care to design our new laboratory experience to foster the “research-like” independence and problem-solving abilities of our students.
Our plan (outlined herein) was to convert our traditional “verification-type” labs into project-oriented labs in which students receive general guidelines and purposes for the experiment, not detailed “cookbook” instructions. The project in the first quarter is to isolate and purify egg-white lysozyme; in the second quarter students focus on determining the kinetic parameters of the enzyme acid phosphatase. In keeping with our desire to remove the verification-type lab (where students learn a principle in lecture and then test it in the lab) from our curriculum, we ask students to buy a lab text (11), which they are instructed to use to guide them through the theory and practice of the required laboratory techniques. That is not to imply that we do not cover the necessary theory (chromatography, enzyme assays, electrophoresis, etc.) in the lecture. The material is carefully covered in the classroom; however, lecture courses are designed to present a coherent, modern view of the chemistry of biomolecules without the pressure to complete a certain topic before the students encounter it in the lab. The usual scenario is that students have been introduced to the theory of a specific technique in lecture well before it is needed in the laboratory. In our limited experience, we have found that the project labs have greatly increased our students’ academic maturity, independence, and sense of accomplishment. Lysozyme from Egg Whites The first-quarter lab is based on the purification of lysozyme as done by Hurst, Keenan, and Son (12). A slight experimental modification involving use of dialysis instead of filtration to purify the egg white prior to chromatography has been incorporated and is carried out as follows. The egg whites are dialyzed against 0.10 M sodium acetate buffer, pH 4.5, and then centrifuged for 20 min at 10,000 rpm. Five milliliters of supernatant is diluted with 15 mL of 0.050 M Tris, 0.050 M NaCl, pH 8.2, after which it is ready for chromatography. The chief modification in our new method is the approach students take to the project. They are given an overall procedure, but not specific volumes or amounts of buffers or solutions to pipet or measure. They start out calculating buffer concentrations and preparing these buffers themselves, as in a real laboratory situation. In addition, it is left to them to “figure out” how to pack and run a column. The professor takes an active role as an “expert research assistant.” The students are given the entire quarter to complete the isolation and purification project, but most of them finish long before the “end-of-the-quarter rush”.
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In the Laboratory
Determination of the Kinetic Parameters of Acid Phosphatase The approach to the second-quarter lab is similar. This project is based on determining the kinetic parameters of acid phosphatase. The experimental protocols are from Stenesh (13). Students are told the appropriate buffer concentrations, but must make up their own solutions and determine the appropriate volumes. They are told to determine (i) the K m and Vmax of the enzyme, (ii) whether phosphate is an inhibitor and if so what type, (iii) the KI (if appropriate), and (iv) the temperature and pH optima of the enzyme. As in the first quarter, students are required to work out their own experimental protocols to accomplish these goals. In this part of the laboratory program, they learn to apply the graphical methods discussed much earlier in lecture to determine kinetic parameters. This direct application of lecture to a non-verification-type lab is an experience that most students find rewarding and intellectually stimulating. This reinforces our belief that the design of the new laboratory program serves to clarify the data–theory (or “principle–practice”) connection that is often very difficult for undergraduates to grasp. Summary Students are given one quarter to accomplish each project. Most students work hard to finish the project early (before finals start) and then find time to write an unrushed lab report. We further encourage this efficient use of time by allowing students to turn in rough drafts of their lab reports for professorial editing. This critiquing usually produces a much more detailed final report. Previously, we found that students were often long on experimental details and short on overall concept, such as what gels and columns are actually supposed to ac-
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complish for the experimenter. However, the final reports of the new project-type labs revealed that students had a much better grasp of the concepts than with our earlier “cookbook” type labs. Overall, students greatly enjoyed the extra freedom granted them and quickly learned how to work independently in a laboratory setting. They were also forced to develop overall strategies for the entire project, such as budgeting time wisely and making sure that all reagents were ready before the extract was added to the chromatography column. The only other labs in our curriculum that require students to plan for an entire quarter as opposed to one afternoon at a time are qualitative organic analysis and general qualitative analysis. We feel certain that it is no coincidence that students enjoy those labs and often comment that these (along with the “new” biochemistry labs) are the toughest but most meaningful lab experiences of their undergraduate careers. Literature Cited 1. Kenigsberg, P. A.; Blanke, S.R.; Hager, L. P. J. Chem. Educ. 1990, 67, 177–178. 2. Keller, J. W.; Wise, M. K.; Reynolds, R. A. J. Chem. Educ. 1991, 68, 265–266. 3. Boyer, R. F. J. Chem. Educ. 1991, 68, 430–432. 4. Farrell, S. O.; Farrell, L. E.; Dircks, L. K. J. Chem. Educ. 1991, 68, 707–709. 5. Clapp, C. H.; Swan, J. S.; Poechmann, J. L. J. Chem. Educ. 1992, 69, A122–A126. 6. Mostad, S. B.; Glasfeld, A. J. Chem. Educ. 1993, 70, 504–506. 7. Utecht, R. E. J. Chem. Educ. 1994, 71, 436–437. 8. Giuliano, K. A. J. Chem. Educ. 1994, 71, 590–591. 9. Farrell, S. O. J. Chem. Educ. 1994, 71, 1095–1096. 10. Burgess, S. K. J. Chem. Educ. 1991, 68, 46–47. 11. Robyt, J. F.; White, B. J. Biochemical Techniques: Theory and Practice; Waveland Press: Prospect Heights, IL, 1987. 12. Hurst, M. O.; Keenan, M. V.; Son, C. C. J. Chem. Educ. 1992, 69, 850–851. 13. Stenesh, J. Experimental Biochemistry; Allyn and Bacon: Newton, MA, 1984; p 181.
Journal of Chemical Education • Vol. 74 No. 2 February 1997
