In the Laboratory
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A Structure–Activity Investigation of Photosynthetic Electron Transport An Interdisciplinary Experiment for the First-Year Laboratory Kerry K. Karukstis,* Gerald R. Van Hecke, Katherine A. Roth, and Matthew A. Burden Department of Chemistry, Harvey Mudd College, Claremont, CA 91711; *
[email protected] Rationale for the Experiment Many exciting research questions lie at the intersection of the disciplines of chemistry, biology, and physics. Through a recent curricular innovation at Harvey Mudd College, we engage first-year students in an introductory laboratory course that illustrates the interdisciplinary nature of science today. As recommended by the National Science Foundation in a recent review of undergraduate education (1), our Interdisciplinary Laboratory or “ID Lab” (2) seeks to “build inquiry, a sense of wonder and the excitement of discovery” (1) through experiments that involve an investigative style of laboratory more representative of research. In designing our ID Lab, we sought experiments that blend the fields of chemistry, biology, and physics and emphasize question and hypothesis formulation and testing. We naturally looked toward the research programs of the Harvey Mudd College faculty as sources of ideas. One ID Lab experiment that developed from faculty research interests (3–6 ) uses the process of photosynthesis to illustrate an interdisciplinary approach. Students use absorption spectroscopy to monitor the rate of the initial steps of plant photosynthesis. These light-activated steps involve electron transfer between specialized molecules in chloroplasts, the cell organelles responsible for photosynthesis. Students measure the effect of several inhibitors (i.e., herbicides) on the electron transfer rate and formulate a hypothesis to test the relationship between inhibitor activity and inhibitor structure. This application of a physical technique to monitor the rate of a chemical process in a biological system aptly illustrates the interdisciplinary approach. Curricular Placement and Connections The ID Lab is part of our required first-year curriculum in which all students take one year of general chemistry lecture and laboratory and one year of introductory physics lecture with a one-semester laboratory. Students who elect to enroll in the ID Lab substitute this course for the fall chemistry laboratory and the spring physics laboratory. The ID Lab was offered in the 1999–2000, 2000–2001, and 2001–2002 academic years. This experiment involves multiple chemistry concepts including reaction rate determination, electron transfer in oxidation–reduction reactions, concentration quantification from physical properties, relationship of absorbed wavelength to observed color, and dependence of intermolecular forces on chemical structure. As these topics are generally covered in the second semester of our general chemistry lecture course, the electron transport experiment occurs in the spring-semester ID Lab. Cognizant that most first-year students are not concurrently enrolled in our introductory biology course, we
provide background material in the laboratory manual on the overall process of photosynthesis, the structure and preparation of plant chloroplasts, and the electron transport process. To conduct the experiment successfully, however, only an understanding of the essential aspects of photosynthesis and the spectroscopic assay employed in the experiment are necessary, and we review these concepts briefly at the first laboratory meeting before beginning laboratory work. A concise overview of quinone nomenclature and structure also enables students to easily consider chemical structure in designing their experimental hypotheses. Overview of Experimental Procedures and Techniques Learned Students test the effectiveness of substituted quinones (Table 1) to act as inhibitors of photosynthetic electron transport in spinach chloroplasts. The spectroscopic assay to measure the electron transport rate involves the addition of dichloroindophenol (DCIP) to the chloroplast sample. DCIP positions itself as a terminal electron acceptor in the photosystem II electron transfer chain (see Fig. 1A) (7). In aqueous solution, oxidized DCIP appears blue (absorbing light maxiTable 1. Suggested Substituted Quinones to Test as Potential Electron Transport Inhibitors O 2
3
1 4
O 6
2
5
3
O 8
1 4
7
2
6
3
5
O
1
O 8
9 10 4
O
7
2
6
3
Name and Shorthand Designation
8
10
5
4
5
O
9 1
7
6
O
CAS Registry No.
Suggested Supplier
1,4-Benzoquinone (1,4-BQ)
106-51-4
Aldrich
Tetramethyl-1,4-ylbenzoquinone (2,3,5,6-tetra-CH3-BQ; duroquinone)
527-17-3
Aldrich
Tetrahydroxy-1,4-benzoquinone (2,3,5,6-tetra-OH-BQ)
123334-16-7 Aldrich
1,4-Naphthoquinone (1,4-NQ)
130-15-4
Pfaltz & Bauer
2-Methyl-1,4-naphthoquinone (2-CH3-NQ)
58-27-5
Aldrich
2-Hydroxy-1,4-naphthoquinone (2-OH-NQ)
83-72-7
Pfaltz & Bauer
5-Hydroxy-1,4-naphthoquinone (5-OH-NQ)
481-39-0
Pfaltz & Bauer
9,10-Anthraquinone (AQ)
84-65-1
Aldrich
1-Hydroxyanthraquinone (1-OH-AQ)
129-43-1
TCI
1,4-Dihydroxy-anthraquinone (1,4-diOH-AQ, quinizarin)
81-64-1
Aldrich
2-Ethyl-anthraquinone (2-CH2CH3-AQ)
84-51-5
Aldrich
1,4-Anthraquinone (1,4-AQ)
635-12-1
Lancaster
NOTE: The chemical structure and numbering system is illustrated from left to right for 1,4-benzoquinones, 1,4-naphthoquinones, 9,10-anthraquinones, and 1,4-anthraquinones.
JChemEd.chem.wisc.edu • Vol. 79 No. 8 August 2002 • Journal of Chemical Education
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In the Laboratory −
0.7
A1
− A2
A3 DCIP −
A1 A2 A3 DCIP
A
−
A1 A2 A3 DCIP
Q A1 A2 A3 DCIP
B
A1 A2 Q DCIP
Absorbance at 600 nm
A1 A2 A3 DCIP
0.6
0.5
0.4
0.3
A3
0
20
40
60
80
100
120
140
160
Time / s Figure 2. Rate of change in absorbance of DCIP at 600 nm upon illumination of spinach chloroplasts with no added quinones.
−
A1 A2 Q DCIP −
A1 A2 Q DCIP −
C
A1 A2 Q DCIP A1 A2 Q DCIP
Figure 1. A: The added dye DCIP positions itself as a terminal electron acceptor in the chain of electron acceptors (A1, A2, A3, …) in photosystem II. B: Substituted quinones displace one of the naturally occurring electron acceptors (here designated A3). C: Electron transfer to DCIP is hindered by those quinones Q that act as strong inhibitors of photosynthetic electron transport.
mally at 600 nm), but it is colorless in its reduced state. Thus, the rate of absorbance decrease at 600 nm is proportional to the rate of electron transfer. Substituted quinones bind to an herbicide-binding protein and displace one of the naturally occurring electron acceptors in the electron transport chain (Fig. 1B). Students use a molecular modeling program on laptop computers to visualize the quinone structural aspects that promote binding and thereby hinder electron transport (Fig. 1C).1 By correlating quinone structure with inhibitory activity, a model for the herbicide-binding region in plant chloroplasts is devised. Equipment Required student equipment includes micropipettors (e.g., 20 µL, 100 µL, and 5 mL capacity), timer, a small pen flashlight as a light source, acrylic cuvettes, cuvette rack, quartz cuvette, small funnel, filter paper (No. 1, 90 mm), Kimwipes. A UV–vis spectrophotometer is required; temperature control is desirable but not necessary. Chloroplast preparation (not by the student) requires a high-speed centrifuge capable of spins at 6000g. Chemicals Table 1 lists substituted quinones of various ring sizes to test as inhibitors. A commercial herbicide, DCMU (diuron, 3-(3,4-dichlorophenyl)-1,1-dimethylurea), is also tested for comparison. Spinach chloroplasts are prepared and suspended in a buffer solution composed of sucrose, HEPES (N-(2hydroxyethyl)piperazine-N1-(2-ethanesulfonic acid), and sodium chloride at pH 7.5. An 80% (v/v) acetone-in-water solution is needed to determine the chlorophyll concentration 986
O
−
C N
hydrophillic region
H OH
O
HO
possible hydrophillic region
hydrophobic region CH3 CH2
OH
O
Figure 3. A hypothetical quinone is drawn with the structural features predicted to lead to maximal inhibition of electron transport. On the basis of this “ideal” inhibitor of photosynthetic electron transport, a model of the herbicide binding region is sketched defining the dimensions and hydrophobic and hydrophilic character of this binding region. Hydrogen bonding of the quinone to an amide linkage is one noncovalent interaction responsible for the binding of the inhibitor to the herbicide-binding protein.
of the chloroplasts. Dichloroindophenol (DCIP) is used for the spectroscopic assay. Hazards The spinach chloroplast and buffer solutions present no health or environmental hazard and may therefore be disposed of by washing down the sink. The quinone inhibitors are generally not water soluble at high concentration and are dissolved in an organic solvent to form the stock solutions. Chloroplast samples with quinone inhibitors and DCIP should be disposed of in halogenated organic waste containers. Sample Student Results Figure 2 is a graph of DCIP absorbance at 600 nm as a function of time for spinach chloroplasts with no added quinones. Since absorbance decreases owing to DCIP photoreduction, the slope of the line is proportional to the rate of photosynthetic electron transport. Quinone inhibitory strength is ranked by calculating the ratio of the slope of each
Journal of Chemical Education • Vol. 79 No. 8 August 2002 • JChemEd.chem.wisc.edu
In the Laboratory Table 2. Ranking of Observed Quinone Inhibitory Strength for Selected Quinones Ratio =
Rate of Inhibited Electron Transport Rate of Uninhibited Electron Transport
Substituted Quinone
Table 3. Students' Post-Experiment Assessment of the Most and Least Enjoyable Aspects of the Experiment Response (%)
Aspect of the Experiment Most Enjoyable
Pooled Class Average
Determined by a Student Pair
2000–2001
1999–2000
Use of the spectrometers and micropipettors
36
2-CH3-NQ
0.20 ± 0.01
0.19 ± 0.06
0.17 ± 0.09
Data analysis
23
1-OH-AQ
0.29 ± 0.01
0.20 ± 0.05
0.22 ± 0.10
Working with and learning about the quinone inhibitors
23
1,4-diOH-AQ
0.32 ± 0.02
0.24 ± 0.07
0.34 ± 0.10
Molecular modeling of the quinone inhibitors
16
1,4-AQ
0.37 ± 0.06
0.32 ± 0.07
0.47 ± 0.07
1,4-NQ
0.60 ± 0.01
0.53 ± 0.12
0.55 ± 0.10
Learning about photosynthesis and plant chloroplasts
2-CH2CH3-AQ
0.60 ± 0.02
0.54 ± 0.11
0.47 ± 0.10
5-OH-NQ
0.63 ± 0.03
0.62 ± 0.12
0.67 ± 0.08
1,4-BQ
0.69 ± 0.02
0.67 ± 0.07
0.76 ± 0.11
2-OH-NQ
0.76 ± 0.07
0.69 ± 0.12
0.76 ± 0.14
2,3,5,6-tetraCH3-BQ
0.76 ± 0.03
0.69 ± 0.09
0.85 ± 0.13
9,10-AQ
0.84 ± 0.07
0.78 ± 0.17
0.86 ± 0.11
2,3,5,6-tetraOH-BQ
0.94 ± 0.02
0.84 ± 0.19
0.93 ± 0.07
DCMU a
0.043 ± 0.005
—
—
NOTE: The ability of a quinone to inhibit electron transport is determined by the ratio of the rate of change in DCIP absorbance at 600 nm for quinone-treated chloroplasts with respect to the observed rate in untreated chloroplasts. Effective electron-transport inhibitors yield ratios close to zero; quinones with no ability to bind to the herbicide-binding region have no effect on electron transport rate and yield ratios close to 1. a The commercial herbicide known as diuron or 3-(3,4-dichlorophenyl)-1,1-dimethylurea.
inhibited trial to the uninhibited rate (Table 2). Figure 3 illustrates the quinone structural features proposed for optimal ability to block electron transport and depicts a model of the proposed herbicide binding region. Assessment In post-experiment evaluations, 97% of the students identified various contributions from two or three disciplines. This demonstrates a successful illustration of interdisciplinarity. Specifically, students recognized that a physical technique (spectroscopy or light absorption) could be used to characterize chemical reactions (noted as electron transport, quinone inhibition, inhibitor binding, or DCIP reduction) in a biological system (chloroplasts) or process (photosynthesis). Over 90% of the students felt that the interdisciplinary nature of the investigation was clearly illustrated. The rest indicated a desire to prepare the chloroplasts themselves or to use different types of plant material (a reasonable pedagogical request, but impractical owing to time and equipment constraints). On a scale of 1 (not improved) to 5 (greatly improved), students felt that the investigation significantly enhanced their skills in analyzing and interpreting data (4.0), strongly improved their ability to collect and record scientific data (3.7) and to formulate and test a scientific question (3.5), and modestly promoted their ability to work with a partner (3.3) and communicate scientific results to others (3.1). Further comments summarized in Table 3 indicate that students valued the use of specialized equipment and chemicals (their first exposure to UV–vis spectrometers and micropipettors), the analysis of an extended data set, and the
2
Least Enjoyable Repetition and/or time required to collect the data
58
Analysis of the results
20
Lack of self-preparation for the experiment
16
Variability of results
3
Use of spectrometer
3
introduction to molecular modeling. As an array of results is required to test a specific hypothesis, it is understandable that many students disliked the repetition or time required for data collection. However, no student needed more than the four-hour laboratory period to complete the data acquisition. Interestingly, equal proportions of students found the data analysis to be the most (23%) and least (20%) enjoyable aspect of the experiment. Some students indicated that they had not prepared adequately for the experiment, and we suspect that their modest preparation affected both the time required to complete the experiment and their understanding of the data analysis. To modify the experiment in the future, students suggested no changes (14%), shortening the experiment (29%) or providing more time (7%), improving self-preparation (36%), and working with different plants (7%) or another class of herbicides (7%). In response to our spring 2000 assessment, in spring 2001 we reduced the number of quinones to be tested from 16 to 12. In the next offering, students will select an appropriate subset of 6–10 quinones to test their hypothesis. They will be reminded that a convincing test of their hypothesis will demonstrate structural features that lead to both pronounced and insignificant inhibitory activity. Curricular Successes of the Experiment In our experience this investigation provides an innovative kinetic and spectroscopic analysis that engages students through its interdisciplinary nature, investigative style, and real-world application. Students enjoy formulating and testing a hypothesis relating quinone structure and chemical activity. This commitment to the experiment’s outcome improves the quality of data collection and mitigates the repetitive nature of the measurements. The experiment provides opportunities to work with a functioning plant organelle, broadening the students’ conception of biological systems, and with molecular modeling software, a practice not common at the introductory level. The connection between microscopic parameters and macroscopic behavior
JChemEd.chem.wisc.edu • Vol. 79 No. 8 August 2002 • Journal of Chemical Education
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In the Laboratory
is also aptly illustrated. The interpretation of results to model the herbicide-binding site is an extension of the study typical of research scientists but too infrequently experienced by undergraduates, especially in introductory courses. The derivation of the experiment from a faculty research program also adds to the student appeal. Acknowledgments This effort was funded by a National Science Foundation Award for the Integration of Research and Education (AIRE) to Harvey Mudd College. We thank the Harvey Mudd undergraduates who have participated in the Interdisciplinary Laboratory. Their interest, enthusiasm, and constructive comments have contributed immensely to the success of this educational venture. We gratefully acknowledge the assistance of Karen A. Yoshino, Executive Assistant to the President for Institutional Research and Assessment at Harvey Mudd College. W
Supplemental Material
Complete student experimental procedures, instructions for using the software Molecular Modeling Pro, prelab assignments with solutions, instructions for isolating the chloroplasts and preparing indicator and inhibitor solutions, a sample grade sheet, and sample data for illustrating reaction rate determination are available in this issue of JCE Online. Note 1. The current version of this PC-based software, Molecular Modeling Pro, is available for WIN 95/98 and WIN NT systems
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from Chemistry SW, Inc., Fairfield, CA. The single-user educational price is $149. Any software that computes interatomic distances may be substituted; calculations using standard bond lengths and angles would also generate the information needed to characterize the dimensions of the binding site.
Literature Cited Undergraduate authors are designated by an asterisk (*). 1. Committee for the Review to the National Science Foundation Directorate for Education and Human Resources. Shaping the Future: New Expectations for Undergraduate Education in Science, Mathematics, Engineering, and Technology; Executive Summary; http://www.ehr.nsf.gov/ehr/due/documents/review/ 96139/summary.htm (accessed Mar 2002); Executive Summary: NSF 96-141; Report: NSF 96–139. 2. Van Hecke, G. R.; Karukstis, K. K.; Haskell, R. C.; McFadden, C. S.; Wettack, F. S. J. Chem. Educ., in press. 3. Karukstis, K. K.; Boegeman, S. C.;* Fruetel, J. A.;* Gruber, S. M.;* Terris, M. H.* Biochim. Biophys. Acta 1987, 891, 96–139. 4. Karukstis, K. K.; Gruber, S. M.;* Fruetel, J. A.;* Boegeman, S. C.* Biochim. Biophys. Acta 1988, 932, 84–90. 5. Karukstis, K. K.; Berliner, M. A.;* Jewell, C. J.;* Kuwata, K. T.* In Current Research in Photosynthesis, Vol. I; Baltscheffsky, M., Ed.; Martinus Nijhoff: Dordrecht, 1990; pp 96–139. 6. Karukstis, K. K.; Berliner, M. A.;* Jewell, C. J.;* Kuwata, K. T.* Biochim. Biophys. Acta 1990, 1020, 96–139. 7. Hall, D. O.; Rao, K. K. Photosynthesis, 4th ed.; Edward Arnold: London, 1987.
Journal of Chemical Education • Vol. 79 No. 8 August 2002 • JChemEd.chem.wisc.edu
