In the Laboratory
Equilibrium Gel Filtration Chromatography for the Measurement of Protein–Ligand Binding in the Undergraduate Biochemistry Laboratory
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Douglas B. Craig Department of Chemistry, University of Winnipeg, Winnipeg, MB, Canada R3B 2E9;
[email protected] Teaching laboratory exercises involving gel filtration chromatography have generally focused on protein purification or size estimation, using both “wet” experiments (1–3) and computer simulations (4, 5). One application of gel filtration chromatography, referred to as equilibrium gel filtration chromatography, has been used to quantitate ligand binding to a protein (6–7). The binding of ATP to cytochrome c is examined in this experiment. Cytochrome c has been shown to bind up to three molecules of ATP under low ionic strength conditions. Two of these binding sites are of low affinity and do not bind ATP under cellular conditions. It is the third site that is of biological significance. This binding site is conserved throughout the evolutionary tree and is functional under cellular conditions. The saturation of the binding responds to the energy status of the cell and results in the inhibition of the activity of cytochrome c (8–11). The cytochrome c–ATP experiment was designed to be completed by senior undergraduate students in a three-hour period. It has been used at the University of Winnipeg for three academic years and was designed to complement lectures on liquid chromatography and the use of spectroscopy in the measurement of proteins. However this experiment is also a useful tool in the teaching of additional topics including dynamic equilibrium, liquid flow, and kinetic parameters. Experiment
Overview A gel filtration column was equilibrated with buffer containing ATP at the desired concentration. Cytochrome c was dissolved in the identical buffer. The cytochrome c was loaded onto the column and the same ATP-containing buffer used as the mobile phase. A portion of the ATP in the solution in which the cytochrome c was dissolved became bound to the cytochrome c. This portion of ATP, being associated with the cytochrome c, moved faster down the column than the ATP that remained unbound. This resulted in the depletion of ATP from the fractions in the elution profile corresponding to the loaded solution and its appearance in the fractions in which the cytochrome c eluted. The chromatogram therefore appeared as a baseline signal of the ATP with a peak, owing to the ATP bound to the cytochrome c, followed by a trough, owing to the decrease in ATP in the loaded solution (9, 10, 12). Measurement of ligand binding by equilibrium gel filtration chromatography requires determination of the concentration of both protein and ligand in each fraction. For this reason cytochrome c was chosen for this experiment. The absorbance spectra of most proteins overlap extensively with 96
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ATP. However the spectrum of cytochrome c, a heme-containing protein, extends into the visible range whereas the spectrum of ATP does not. Quantitation of both species can be achieved by monitoring the absorbances at 260 and 410 nm: cytochrome c only absorbs at the latter wavelength, while both cytochrome c and ATP absorb at 260 nm. Based on the A260兾A410 ratio of the protein, the absorbance due to ATP at 260 nm and its concentration can be calculated. An additional advantage is the commercial availability and low cost of both ATP and cytochrome c. Since ATP binding to cytochrome c occurs regardless of the source of the protein, the bovine protein was chosen as it is least expensive.
Materials The buffer was filter-sterilized 25 mM HEPES (pH 7.5) containing 100 mM NaCl and approximately 0.5 mM ATP. A 1 mM cytochrome c solution was made by addition of powdered bovine heart cytochrome c to 2 mL of buffer. It is essential that the cytochrome c be dissolved in the same buffer as that used for the gel filtration. An additional solution of approximately 10 µM cytochrome c was made in 25 mM HEPES (pH 7.5) buffer containing no ATP. All chemicals were obtained from Sigma (St. Louis, MO). A Gradifrac (with P-1 Pump) benchtop low-pressure liquid chromatography system (Amersham Pharmacia Biotech, Baie d’Urfe, PQ) was used in the teaching laboratory. The system was set up with a 1-mL injection loop. The chromatography column was 60-cm long and had a 1-cm internal diameter. The column was packed with Sephadex G-25F (Amersham Pharmacia Biotech) and equilibrated by flushing with 200 mL of HEPES兾NaCl兾ATP buffer at a flow rate of 1 mL min᎑1. Absorbances were measured using a Hewlett Packard 8452A diode array spectrophotometer (Palo Alto, CA). Procedure One milliliter of 1 mM cytochrome c in the HEPES兾NaCl兾ATP buffer was loaded onto the column and eluted at a flow rate of 1 mL min ᎑1 using the HEPES兾NaCl兾ATP buffer as the mobile phase. Forty 1-mL fractions were collected. Each fraction was diluted 30-fold and the absorbances at 410 and 260 nm were determined. The UV–visible spectra of the cytochrome c solution in HEPES buffer was also measured. Hazards There are no noteworthy hazards to the students or the instructors. No cautions beyond normal laboratory safe practices are required.
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In the Laboratory
Discussion To perform this experiment it is necessary to determine the relative absorbance of cytochrome c at 260 and 410 nm. Rather than giving this ratio to the students, the students were required to obtain it by determining the visible spectrum of the protein. This aspect of the exercise was done to facilitate discussions on the spectra of proteins in general and heme proteins in particular. The elution data collected by a fourth-year undergraduate student performing this experiment in the course are shown in Figure 1. Similar results were obtained by the other students. The first 17 fractions consisted of the elution buffer containing the 0.5 mM ATP. The cytochrome c and the bound ATP eluted in fractions 18–26. The buffer from which the bound ATP was removed eluted in the following fractions followed by a return to the baseline ATP level (see the top curve in Figure 1). ATP binding to cytochrome c is too weak at low concentrations to be detected by this method. The use of relatively high concentrations of both protein and ligand are required. For this reason, on-line detection of the eluent was not performed since its absorbance was far above the usable range. A 15-fold dilution of the fractions was sufficient to reduce the maximum absorbances to approximately 0.8. ATP absorbs at 260 nm but not at 410 nm whereas cytochrome c absorbs at both wavelengths. The fraction of the absorbance at 260 nm corresponding to ATP in a given fraction was calculated by,
A260, ATP = A 260 − A 410
A260,cytc A410, cytc
where the ratio A260,cytc兾A410,cytc was determined from the spectra of the cytochrome c dissolved in HEPES and in the absence of ATP. The concentrations of ATP and cytochrome c were determined from their absorptivity coefficients of 15,400 M᎑1 cm᎑1 at 260 nm and 106,100 M᎑1 cm ᎑1 at 410 nm, respectively. Both absorptivity coefficients were assumed to be unaffected by the binding. The ATP corresponds not only to that which is bound but also to the background amount present in the elution buffer. The concentration of ATP in the elution buffer was determined from the average absorbance of fractions 1–17. The ratio of moles bound ATP to moles cytochrome c was determined from the fractions 18–26. The number of moles of bound ATP should be equivalent to that amount deficient from the later eluting fractions. However the amount of bound ATP calculated from the eluting “ATP trough” is less reliable than that from the “ATP peak”, typically being artifactually lower. This is because commercial preparations of cytochrome c may contain small molecular weight impurities that absorb at 260 nm and co-elute with the trough. Such impurities are readily observed when performing dialysis on cytochrome c. Dialysis of commercial preparations of this protein result in the appearance of a heme color in the surrounding solution whereas dialysis of the further purified protein does not. For the sake of simplicity we utilize commercial preparations of cytochrome c that were not further purified and the data obtained from the “ATP peak”. An optional inclusion to this experiment is the calculation of the average number of ATP molecules bound based on the “ATP trough”. Comparison of this with that obtained with the “ATP” peak will allow the students to comment on the presence of impurities. Results Using the experimentally determined A260兾A410 ratio of 0.20, the average number of ATP molecules bound per cytochrome c was found to be 1.4 under the conditions used, in accordance with expectations. This incorporates specific binding at the arginine91-containing site as well as that at the two weaker sites. This value is not of any particular biological significance. The buffer used, 25 mM HEPES (pH 7.5) containing 100 mM NaCl, is not representative of cellular conditions nor is the concentration of ATP used. However, this buffer was chosen because the binding is such that it can be easily and reproducibly detected at concentrations of both ATP and cytochrome c whereby a single dilution is sufficient to obtain absorbance readings within the usable range at both 260 and 410 nm. Experience has shown that if two separate dilutions were required for each fraction some of the students might have difficulty finishing the laboratory experiment in the allotted time. Conclusion
Figure 1. Absorption versus fraction number of the equilibrium gel filtration liquid chromatography run. The curve at 260 nm results from absorption of both ATP and cytochrome c while the absorption at 410 nm is only from cytochrome c.
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This laboratory exercise has been used in the senior biochemistry course at the University of Winnipeg for three years. It combines liquid chromatography and absorbance spectroscopy and allows the students to produce a quantitative result within a single three-hour period. The benchtop
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In the Laboratory
liquid chromatography system is not necessary as a simple gravity-fed column will suffice, making the experiment very economical. W
Supplemental Material
Instructions for the students and notes for the instructor are available in this issue of JCE Online. Literature Cited 1. Pugh, M. E.; Schultz, E. Biochem. Mol. Biol. Ed. 2002, 30, 179–183. 2. Assis-Pandochi, A. I.; Cspadro, A. C.; Lucisano-Valim, Y. M. Biochem. Ed. 1998, 26, 63–65. 3. Dewhurst, F. J. Chem. Educ. 1969, 46, 864–865.
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4. Cameselle, J. C.; Cabezas, A.; Canales, J.; Jesus Costas, M.; Faraldo, A.; Fernandez, A.; Maria Pinto, R.; Meireles Ribeiro, J. Biochem. Ed. 2000, 28, 148–153. 5. Griffith, T. W. J. Chem. Educ. 1989, 66, 407–408. 6. Hummel, J. P.; Dreyer, W. J. Biochem. Biophys. Acta 1962, 63, 530–532. 7. Wiseman, A.; Azari, M. R. Biochem. Soc. Trans. 1982, 10, 135– 136. 8. Corthesy, B. E.; Wallace, C. J. A. J. Biochem. 1986, 236, 359– 364. 9. Craig, D. B.; Wallace, C. J. A. J. Biochem. 1991, 279, 781– 786. 10. Craig, D. B.; Wallace, C. J. A. Protein Sci. 1993, 2, 966–976. 11. Craig, D. B.; Wallace, C. J. A. Biochemistry 1995, 34, 2686– 2693. 12. Margalit, R.; Schejter, A. Eur. J. Biochem. 1973, 31, 500–505.
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