Biochemistry Laboratory

FTNMR Analysis of the Anaerobic Yeast Metabolism of [1-¹³C] Glucose

Introduction:

Metabolism may be defined as the process by which living organisms acquire and use free energy. In chemotrophs, energy is acquired by the oxidation of organic compounds (catabolism) and used for either motion, to maintain osmotic pressure in the cell, or to build specific molecules via biosynthetic reactions (anabolism). Metabolic pathways for either catabolism or anabolism consist of a series of reactions, with each reaction catalyzed by a specific enzyme.

The glycolysis metabolic pathway is common to almost all living chemotrophic organisms. In this catabolic pathway, glucose is oxidized to pyruvate by a series of ten (10) enzymatic steps.. During the process, high energy phosphate containing compounds are formed. The free energy of these compounds are used to form ATP, the Aenergy currency@ of the cell. For every oxidation process, you must have a corresponding reduction. During glycolysis, NAD⁺ is reduced to NADH. For an anaerobic system, such as yeast in a nitrogen environment, the problem is how to recycle the cells= NADH reserves back to NAD⁺ so that glycolysis may continue. Yeast does this by the two step enzymatic conversion of pyruvate first to acetaldehyde and carbon dioxide. Acetaldehyde is then reduced to ethanol (a process we call fermentation), with the corresponding oxidation of NADH back to NAD⁺ (see handouts for a "visual" on this process).

As we have discussed in the lecture, one of the major ways to investigate metabolic pathways is to use isotopes. This allows scientists to follow and "map out" the various products formed during the course of metabolic enzymatic reactions. Today you will attempt to follow carbon-13 labeled glucose (labeled at the C-1, or anomeric carbon atom) to its ethanolic fate. You will attempt to verify that the labeled carbon has really ended up at the C-2 position of ethanol.

Carbon-13 FTNMR

[1-¹³C] Glucose is a good choice for this investigation. The most abundant isotope (~98 %) of carbon, carbon-12, has a even mass number and an even charge number. By quantum rules, this isotope has a nuclear spin of zero. As a nuclei must have a spin to generate the magnetic vector needed to be "seen" by nuclear magnetic resonance, carbon-12 is "invisible" to NMR. Carbon-13 however, although low in abundance (~1.1 %), has a half-integral spin number, and thus is "visible" to NMR. The low abundance means that a large number of FTNMR scans must be taken and averaged to see the small signal. We can enhance this signal if we use a carbon-13 labeled compound. In this case, a relatively small number of scans will be needed to "see" the signal. Moreover, as there is a lot of different natural abundance carbon-13 signals in the glucose/yeast batch, using a smaller number of scans will allow these signals to remain "lost" in the noise, allowing us to concentrate on the labeled peak(s).

Experimental Section

Note: This laboratory was adopted from a paper by Mega, et al.

1. Prepare the following solutions:

Stock 10 mM Phosphate buffer solution, pH 7: Using a 250-mL Erlenmeyer flask, dissolve 0.35 g of NaH₂PO₄·H₂O in 100 mL of deionized water. Adjust to pH 7.0 with solid NaOH. Parafilm the flask opening.

Labeled ¹³C-glucose solution: Dissolve 0.054 g of [1-¹³C]glucose (99 atom % ¹³C) in 1.00 mL of the stock buffer solution.

Yeast growth medium: Take the remaining stock buffer solution (~ 99 mL) and dissolve into it 5.0 g of unenriched glucose. Incubate in a water bath at 37° C . After the glucose solution comes to the water bath temperature, add 7 g of active yeast (one packet). Stir occasionally by swirling the flask. The yeast should be allowed to undergo aerobic metabolism for about 15 minutes before preparing the labeled reaction mixture.

Labeled reaction mixture: Transfer a 0.4 mL aliquot of the labeled glucose solution to an NMR tube and deoxygenate the aliquot with a nitrogen purge. A long stainless steel needle is used to accomplish this, and the NMR tube should be suspended in the water bath. Carefully adjust the gas flow so that a constant stream of bubbles is obtained, but that solution is not ejected from the tube. Continue the purge for 15 minutes. Then add 0.2 mL of the incubated yeast growth medium and continue the purge for another 10 minutes. After the purge is completed, remove the needle from the tube, cap the tube with a vented cap (a NMR tube cap with a small hole punched through - acts as a pressure relief valve). Wipe the tube dry, then proceed with NMR spectra collection.

2. NMR spectra collection:

Metabolic Study: Glucose consists of two anomeric forms, α and β, which can interconvert. The carbon-13 spectrum of [1-¹³C] labeled glucose can distinguish between these two forms. You should see peaks at 92 and 96 ppm, corresponding to the α and β forms, respectively. Spectra should be taken every 30 minutes. If the yeast is metabolizing the glucose properly, you should observe a reduction in the height of these initial peaks. You should then start to see additional peaks arise in the spectrum as the glucose is converted into metabolic products. Measure carefully the location(s) and relative heights of these peaks.

Spectra analysis (1-D studies): To confirm that one of the metabolic products formed is ethanol, take the carbon-13 spectra of pure ethanol. Which (if any) of the metabolic product carbon-13 NMR peaks corresponds to an ethanol carbon-13 peak? Does the carbon-13 spectra of ethanol allow you to identify the location of the labeled atom on the ethanol molecule?

Take a ¹H spectrum of pure ethanol. Interpret the spectrum (determine which proton peaks correspond with which carbon positions on the ethanol molecule).

Spectra analysis (2-D HETCOR study): Advanced FTNMR techniques allows us to unambiguously relate the easy-to-determine proton spectra to the harder-to-interpret carbon-13 spectra. We will set up and run overnight (it takes some time) a heteronuclear correlation (HETCOR) spectrum of ethanol. Using this spectrum, you should be able to confirm that ethanol was in fact produced by the yeast metabolic process.

Reference:

Mega, T.L.; Carlson, C.B.; Cleary, D.A. J. Chem. Ed. 1997, 74, 1474.