In the Laboratory
Following Glycolysis Using 13C NMR An Experiment Adaptable to Different Undergraduate Levels T. L. Mega Department of Chemistry, Whitworth College, Spokane, WA 99251 C. B. Carlson and D. A. Cleary* Department of Chemistry, Gonzaga University, Spokane, WA 99258 Glycolysis is the most common metabolic pathway in nature. It is presented to students at a variety of levels in biology and chemistry courses at the undergraduate level. The centrality of glycolysis with respect to other pathways and the fact that detailed reaction mechanisms for each step are known makes study of this system an important part of the standard biochemistry course. Chemistry and biology students often have the opportunity to study the pathway in some detail in upper-division biochemistry courses. In a recent article in this Journal, Schultz emphasized the importance of biology in the modern chemistry curriculum and of chemistry in the modern biology curriculum (1). Laboratory investigations of glycolysis often focus on one or a few isolated reactions within the pathway. This is because of the complexity of the process: with ten or more enzymes simultaneously interacting with more than a dozen metabolites and cofactors, detailed experimental analysis of what is happening is difficult. Over the past 5 years we have had success teaching our students about this pathway using 13C NMR spectroscopy. Although the use of 13C NMR spectroscopy does not overcome the complexities of the problem, it does allow one to focus on particular aspects of the reaction pathway. The primary advantages of 13C NMR spectroscopy are its extremely high resolving power and noninvasive nature. Signals corresponding to glycolytic metabolites can be seen increasing or decreasing as the reaction takes place in the NMR tube. For example, during anaerobic glycolysis, signals corresponding to the α- and β-pyranose anomers of glucose decrease in intensity as the signal corresponding to ethanol increases in intensity. The primary disadvantage of this technique is its lack of sensitivity. This problem is overcome to a great extent by the use of 13C-enriched glucose, which is readily available from chemical suppliers. Because relatively small amounts are needed, the cost per student is reasonable.1 We have performed this experiment in a large introductory biology course, a small advanced chemistry course, and an upper-division biochemistry course. In the biology course, students prepared their own samples. Because of the need to lock on the D2 O signal and tune the instrument quickly, it was necessary to have a teaching assistant help the student collect the NMR spectra. In the biochemistry and advanced chemistry courses, students conducted the entire experiment without benefit of a teaching assistant. In this paper, we focus our attention on the experiment as it was performed in the advanced chemistry course. In the past, use of high field NMR spectrometers by undergraduate students would not have been possible because of the complexity of operation. However, modern software permits straightforward instrument control.
*Corresponding author.
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Experimental Section
Glycolysis Reaction Two solutions were used: 13C-glucose stock solution and yeast growth medium. The glucose stock solution was prepared by dissolving 0.034 g of NaH2PO4?H2 O and 0.090 g of [1-13C]glucose (99 atom % 13C) in 7.5 mL of H 2O and 2.5 mL of D2O (99.8 atom % D). The pH was adjusted to 7.0 by adding solid NaOH. The yeast growth medium was prepared by dissolving 3.5 g of NaH2PO4 ?H 2O and 50 g of unenriched glucose in 1 L of H2O. Again, the pH was adjusted to 7.0 with solid NaOH. To begin glycolysis, 100 mL of the yeast growth medium was placed into a 250-mL Erlenmeyer flask incubating in a water bath set to 37 ± 3 °C. After the solution had come to thermal equilibrium, 7 g of active dry yeast (one packet) was added, and the solution was stirred occasionally by swirling the flask. Throughout this period, the yeast cells were metabolizing under aerobic conditions. The reaction produced foam, and the sweet smell associated with fermentation could be detected. While the fermentation was proceeding, a 400-µL aliquot of the labeled glucose solution was added to an NMR tube. This solution was deoxygenated in the NMR tube with an inert gas purge (nitrogen) using a long needle that reached to the bottom of the tube. A low gas flow was used to prevent the glucose solution from being expelled from the NMR tube. After the yeast had reacted for approximately 15 min, a 200-µL aliquot of the reaction mixture was added to the deoxygenated glucose solution already in the NMR tube. The inert gas purge was continued for an additional 5–10 min. Finally, the purge line was withdrawn and the NMR tube capped immediately. The tube was placed in the NMR spectrometer and spectra were collected. Spectroscopic Details To follow glycolysis by 13C NMR, several scans per spectrum were required for a reasonable signal-to-noise ratio. At 20 °C, the reaction was complete in several hours. A spectrum (256 scans/spectrum) was collected every 12 min. Other relevant instrumental parameters were: tip angle = 30° predelay time = 1 s sweep width = 100 ppm 1H
decoupler, on
data points = 16K temperature = 20 °C and 30°C (± 0.1 °C)
For the proton-coupled spectrum recorded at the end of the glycolysis reaction, 4096 scans were collected. The signal-to-noise ratio in the proton-coupled spectrum is less than in the decoupled spectrum.
Journal of Chemical Education • Vol. 74 No. 12 December 1997
In the Laboratory Discussion The 13C NMR of spectrum of [1-13C]glucose consisted of two peaks (96 and 92 ppm) corresponding to the β and α anomers of glucose, respectively: HO
CH2OH O
HO HO
β
OH
HO
CH2OH O
HO HO
α
OH
As the glucose was converted to ethanol and carbon dioxide, the δ = 92 ppm and δ = 96 ppm peaks lost intensity while new peaks appeared. In Figure 1, the time evolution of the 13C NMR peak from α-[1-13C]glucose is shown along with the peak at δ = 17 ppm, which corresponds to the methyl carbon on ethanol. Students confirmed the identity of the δ = 17 ppm peak by collecting a 13 C NMR spectrum of unenriched ethanol (δ = 16.8 and 57.3 ppm). Acquiring proton-coupled carbon spectra (using gated decoupling to improve the signal-to-noise ratio) provided a wealth of information about the nature of the metabolites. One-bond carbon–hydrogen couplings (1JCH) show how many hydrogens are directly attached to the carbon, and two-bond carbon–hydrogen splittings (2JCH) reveal how many protons are on neighboring carbons. Comparing the splitting pattern in the proton-coupled spectrum of the glycolysis final product (Fig. 2) with that obtained from ethanol, students were further convinced that this product was [2-13C]ethanol. The quartet of triplets is consistent with the carbon 2 of ethanol. Close to the ethanol resonance, a quartet of doublets was also observed. Using known compounds, it was determined that this resonance pattern was not from lactate or acetaldehyde, two likely candidates (2). The origin of this species remains under investigation. Another 13C resonance grew in at δ = 63 ppm as glycolysis proceeded. The intensity of this peak was less than that of the ethanol peak. The proton splitting pattern (Fig. 3) was consistent with either CH2 bound to CH2, or CH2 bound to CH; the signal-to-noise ratio was too low to distinguish between these possibilities. According to den Hollander et al. (3), another major end product of glycolysis is [1-13C]glycerol, δ = 63 ppm. An unenriched glycerol sample was used here for confirmation. The glycerol is produced from dihydroxyacetone phosphate. The reverse process, from glycerol to dihydroxyacetone phosphate, also occurs (4). The time dependence of the intensity of the α-glucose resonance at 20 and 30 °C is shown in Figure 4. When students attempted to fit these data to first- or second-order kinetics, they discovered that neither model adequately described the kinetics of this experiment, especially at long reaction times. The same held true when attempting to model the growth of the ethanol peak at δ = 17 ppm. Because the yeast concentration was not constant during this experiment, a straightforward kinetics analysis suitable for undergraduate chemistry was not possible. Nonetheless, some qualitative kinetic features were evident. First, as the temperature of the reaction was increased from 20 to 30 °C, the metabolic rate increased. Second, as the reaction proceeded and the number of yeast cells increased, the rate (measured as the slope of the NMR signal intensity versus time) increased dramatically for a short time until the glucose became depleted. We also observed that the rate of glycolysis depended upon bubbling N2 gas through the sample before spectrum acquisition. This removed oxygen so that glycolysis proceeded under anaerobic conditions. In the experiments of
Figure 1. Time evolution of the α-[1-13C]glucose and [2- 13C]ethanol 13C NMR peaks at 20 °C. Spectra are 12 min apart.
Figure 2. Proton-coupled 13C NMR spectrum at 30 °C of the δ = 17 resonance of the final product of anaerobic glycolysis by yeast. For both the ethanol resonance and the weaker resonance, the large splitting is 125.7 Hz and the small splitting is 2.4 Hz.
Figure 3. Proton-coupled 13C NMR spectrum at 30 °C of the δ = 63 resonance of the final product of anaerobic glycolysis by yeast. The large splitting is 141.6 Hz and the small splitting is 2.4 Hz.
den Hollander et al. (3), the NMR tube was bubbled with 5% CO2 /95% N during acquisition of spectra to maintain the CO2 /HCO3 { ratio during glycolysis. Their experiments proceeded much more rapidly than ours, suggesting that accumulated gases contributed to slowing the overall reaction rate. A more subtle effect involves the relative reaction rates of the two glucose anomers. In Figure 5, the ratio of the 13C-NMR peak amplitude due to the β anomer versus the α anomer is plotted as a function of reaction time at 30 and 20 °C. Because this ratio increased with reaction time, stu-
Vol. 74 No. 12 December 1997 • Journal of Chemical Education
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In the Laboratory
Figure 4. Plot of peak height (δ = 92 ppm) versus time (min) for α-[1- 13C]glucose: (s ) 20 °C; (• ) 30 °C.
dents were led to two possible conclusions: 1. The α anomer reacts faster than the β anomer. 2. The α anomer reacts exclusively, and the conversion β → α is slow compared to the rate at which α reacts. A similar effect was observed at 20 °C, although the ratio did not change as dramatically. The second conclusion is the correct one, and the temperature dependence of the ratio plotted in Figure 5 suggests that the metabolic rate is more sensitive to temperature than is the β → α conversion rate. Conclusions In this laboratory experiment, students use 13C-NMR spectroscopy to follow the kinetics of a biochemical process. The exercise is appropriate for students at different levels of training depending on what aspects of the experiment are emphasized. For introductory biology students, it provides an interesting example of in vitro monitoring of a biological process. For advanced chemistry students, it presents a challenging kinetics and NMR spectral interpretation problem. For biochemistry students, it provides a straightforward example of modern instrumentation being used to elucidate the mechanism of a complicated biochemical pathway. The different levels of applicability are summarized in Table 1. The major features of this experiment are reproducible: the glucose signal decays and the ethanol signal increases. However, depending on the exact strain and vigor of yeast used in the glycolysis, the number and amount of the additional metabolites (all minor concentrations) will vary.
Table 1. Applications of the
13C
NMR Glycolysis Experiment
Course / Level
Application
Introductory Biology
Metabolism Modern instrumentation Basic lab preparations Kinetics (complicated) NMR Equilibrium (multiple) Regioselectivity Enzymology Glycolysis
Physical Chemistry
Biochemistry
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Figure 5. Ratio of peak intensity of β-glucose versus α-glucose as a function of reaction time: (s) 20 °C; (•) 30 °C.
An attractive feature of this experiment is its openended nature. A number of interesting follow-up experiments are possible. For example: 1. Using 13C-enriched glucose whose labeled carbon is in a position other than 1 would allow for additional mapping of the metabolic pathway. For example, if carbon 3 or 4 is labeled, no ethanol peak will result. 2. Using known compounds, some of the other metabolites could be identified. 3. Inhibitors such as EDTA, which would complex metal ions, could be added. In the case of EDTA, the glucose will not react because a metal ion is involved in the first step of the pathway. 4. The negative effect of temperatures greater than 40 °C on the metabolic process could be explored. Acknowledgments We acknowledge helpful discussions with W. F. Ettinger and K. L. Nakamaye in preparing and interpreting this laboratory experiment. We gratefully acknowledge the support of the National Science Foundation, Instrumentation and Laboratory Improvement Program (ILI Grant DUE9152468), and the Murdock Charitable Trust Fund. Note 1. For example, 1.9 mg of [1- 13C]glucose in a 0.7-mL NMR sample volume gives a 70 mM [1- 13C]glucose solution, which is equivalent to a 6.4 M (natural abundance 13 C) glucose solution. Aldrich Chemical Company currently sells 1000 mg of [1-13C]glucose (99 atom % 13C) for $244, which is enough for 100 such experiments; that is, a little over $2 per experiment.
Literature Cited 1. Schultz, E. J. Chem. Educ. 1996, 73, 447. 2. Villee, C. A.; Solomon, E. P.; Maring, C. E.; Martin, D. W.; Berg, L. R.; Davis, P. W. Biology, 2nd ed.; Saunders: Philadelphia, 1985; pp 177–184. 3. den Hollander, J. A.; Brown, T. R.; Ugurbil, K.; Shulman, R. G. Proc. Natl. Acad. Sci. USA 1979, 76, 6096. 4. Voet, D.; Voet, J. G. Biochemistry, 2nd ed.; Wiley: New York, 1995; Chapter 16.
Journal of Chemical Education • Vol. 74 No. 12 December 1997
