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A simple spectrophotometric method for determining proton release from yeast cells
ANALYTICAL
BIOCHEMISTRY
123, 201-204
(1982)
A Simple Spectrophotometric Method for Determining Proton Release from Yeast Cells CARLOSPASCUALAND Centro
National
ARNO~T KOTYK
de Investigaciones CientQicas. La Habana, Cuba, Czechoslovak Academy of Sciences, 142 20 Prague,
and Institute Czechoslovakia
of Microbiology,
Received December 28, 1981 A simple spectrophotometric acid-base indicator method was developed to determine the rate of proton extrusion after addition of a fermentable hexose to a yeast cell suspension. Unlike the conventional pH-electrode measurements the method gives linear response to changes in proton concentration and makes rapid kinetic determinations possible.
Increasing attention has been focused on the electrochemical potential gradient of protons across cell membranes (1). The proton gradient appears to be the source of energy for ATP synthesis, as well as for active transport in cell organelles (2,3), bacteria (4), and yeast (5) and algal (6) plasma membranes. Determination of the magnitude and mechanism of formation of the transmembrane pH gradient has become a major task in the bioenergetics of lower eukaryotes. Under whatever conditions a change in pH takes place it will depend on the relative buffering capacities of the internal and external media. Changes in intracellular pH are usually monitored with a pH-sensitive glass electrode (5,7,8). However, the time course of changes of actual concentration of H+ ions is less evident since it must be deduced from the logarithmic record. Likewise, small changes in H+ concentration may be difficult to assess. In the present work, a simple indicator method has been used to overcome these shortcomings of the now standard pH estimations. MATERIALS
spectrophotometrically the rate of acid produced when yeast cells are incubated with a fermentable hexose. Using the acidic peak of the spectrum at 540 nm (see Fig. 1) we obtained a linear relationship between addition of protons to methyl red solution and absorbance. For reactions where proton liberation occurs at pH not very far from the pK of the indicator (5.0 for methyl red) the change in absorbance of the dye becomes a quantitative measure of proton concentration. (It was checked that the approximately 0.6 mM methyl red solution had no adverse effect on cell fermentation or respiration, as well as on intracellular pH; for the method see Ref (9).) Strain and culture conditions. The strain 196-2 (a-his-6) of Saccharomyces cerevisiae was used for the study. Cells were grown on minimal medium containing 2% glucose as described previously (10) and were harvested at the end of the exponential phase. The cells were washed with deionized water at 4’C. Preparation of indicator solution. The indicator solution was prepared by dissolving 100 mg methyl red in 20 ml of 20 mM NaOH and diluting to 500 ml with deionized water. It is important that any excess of NaOH in the indicator solution be neutralized with HCl. The amount of acid added for neu-
AND METHODS
Principle of the method. It is based on the use of methyl red as an indicator to measure 201
0003-2697/82/090201-04$02.00/0 Copyright 0 1982 by Academic Press, Inc. All rights of reproduction in any form reserved.
202
PASCUAL
AND KOTYK
WAVELENGTH
( nm 1
I. Absorption spectrum of the acidic and basic forms of methyl red. Methyl red solution (27 0.2 M HCI (acidic form) and in 0.2 M NaOH (basic form) was placed in a cuvette of I-cm light path. The absorption spectrum was registered at a scanning speed of 4 nm s-’ and a recording velocity of 0.2 cm s-‘. FIG. PM) in
tralization is determined by placing a certain volume of the indicator solution in a spectrophotometric cuvette and adding 20-~1 portions of 0.1 M HCl until a change of ab-
sorbance at 540 nm occurs. The amount of HCl added just before this change takes place is used for calculating the amount needed for neutralizing excess NaOH in the 500-ml stock solution. A calibration curve is then used to determine the value of absorbance which corresponds to a given quantity of protons. Usually a change of absorbance of 0.055 at 540 nm corresponds to 10 nmol of protons released in the reaction. Measurement of proton concentration changes. Yeast cell suspension (50 ~1,at 40-
I 20
nmol
H
2. Changes in indicator absorbance due to proton liberation from yeast cells after addition of different sugars. Fifty microliters of a yeast suspension containing 42.7 mg fresh weight per milliliter was added to 2.5 ml of the indicator solution. At the times indicated by the arrows 4 pmol of the sugar was added. The velocity was recorded at a chart speed of 1 cm s-’ with scale expansion of 2.0 absorbance units. FIG.
80 mg/ml) is added to a l-cm cuvette containing 2.5 ml of methyl red solution. After equilibration, 50 ~1 of a specified sugar concentration is added and changes of absorbance are recorded at 540 nm at 30°C in a spectrophotometer (Pye-Unicam SP 1800 was used here). Glucose fermentation was measured by conventional manometric techniques (e.g., Ref. (11)) under the same conditions as the measurements of proton concentration. RESULTS
Figure 2 shows a typical record of absorbance obtained on adding various sugars to
DETERMINATION
OF PROTON
S. cerevisiae. No reaction can be detected if either substrate or cells are excluded from the reaction mixture. The rate of proton release as a function of cell density is linear up to about 6 mg cells in the reaction mixture (Fig. 3). Using higher cell concentrations for the measurement may result in underestimates, apparently due to the generation of buffering capacity by cells in the external medium (12). Proton release rates at different concentrations of glucose follow typical MichaelisMenten kinetics, with highly satisfactory linearity (correlation coefficient r = O-99), yielding a K,,, of 6.4 mM and I/ of 82 pmol min-’ (g yeast)-‘. Manometric measurements of fermentation rates at different concentrations of glucose using yeast cells under the same conditions yield rather similar parameters: Km = 4.8 mM, I/ = 62 pmol min-’ (g yeast)-‘. Addition of mannose which is metabolized more slowly than glucose elicits a 60% response in proton release while addition of nonmetabolizable sugars, such as D-xylose, D-arabinose (13), and D-galactose, prior to induction ( 14) causes no proton release. Addition of carbonylcyanide m-chlorophenylhydrazone as a proton conductor to shortcircuit the pH difference across the cell membrane had an immediate inhibitory effect on the proton release. DISCUSSION
Spectrophotometric measurement of the velocity of proton liberation by means of an indicator has several advantages as the reaction rate is constant and therefore direct calculations can be made. Moreover, the method is suitable for rapid kinetic measurements since it obviates the use of a pH electrode which generally has a slower response than a spectrophotometer. The detection of proton amounts as low as 10 nmol in 2.5 ml of suspension makes the method quite sensitive. The rate of proton liberation in the presence of 1.6 mM glucose observed here was
RELEASE
FROM
203
CELLS
009E +' 006. I
1
0
25
I
5.0 mg cells
I
75
FOG. 3. Rate of proton extrusion as function of cell concentration. Different amounts of yeast cells from a 77 mg/ml suspension were added to 2.5 ml of methyl red solution. The reaction was started after the addition of 2 pmol glucose. The velocity was registered as indicated in Fig. 2.
23 pmol min-’ (g yeast)-‘. Serrano (5) reported 15 to 30 pmol min-’ (g yeast))’ by measuring pH in yeast suspension in the presence of 25 mM glucose which, in extrapolation, would yield approximately 60 pmol min-’ (g yeast))’ by our method. This difference may be considered to be well within the spread of experimental values found with different batches of yeast. Proton extrusion appears to be linked to glycolysis even if there is no single proton pump responsible for this process (15). In fact, production of C02, release of some organic acids (succinic, malic, lactic), K+-H+ exchange, as well as an ATPase-type proton pump appear to be involved. Still, it is easily seen that nonfermentable sugars cause no proton extrusion from yeast cells. Likewise, JimCnez et al. (16) reported that glycolytic mutants of yeast even in the presence of metabolizable sugars are unable to acidify the medium due to the enzymatic defect, Further evidence to support this link lies in the fact that the K,,, value for glucose which causes proton extrusion is very similar to the K,,, for glucose fermentation. The proposed method could be used not only to study various kinetic parameters of the proton liberation process in yeast but also to quantify in a rapid and simple manner the degree of sugar fermentation.
PASCUAL
204 REFERENCES
1. Fillingame, R. H. (1980) Annu. Rev. Biochem. 49, 1079-1113. 2. Mitchell, P. (1961) Nature (London) 191, 144-148. 3. Whatley, F. R. (1975) in Energy Transformation in Biological Systems (Ciba Foundation Symposium), Vol. 31, pp. 41-61, Associated Scientitic Pub., Amsterdam. 4. Oesterhelt, D. (1975) in Energy Transformation in Biological Systems (Ciba Foundation Symposium), Vol. 31, pp. 147-165, Associated Scientific Pub., Amsterdam. 5. Serrano, R. (1980) Eur. J. Biochem. 105,419-424. 6. Komor, E. (1973) FEBS Lett. 38, 16-18. 7. Sigler, K., Knotkova, A., and Kotyk, A. (1978) Folia
Microbial.
23, 409-422.
8. Mitchell, P., and Moyle, J. (1965) Nature 208, 147-151.
(London)
AND KOTYK 9. Slavik, J, (1982) FEBS Left. 140, 22-26. 10. Herrera, L. S., Pascual, C., and Alvarez, X. (1976) Mol.
Gen. Genet.
144, 223-230.
1I. Umbreit, W. W., Burris, R. H., and Stauffer, J. F. (1957) Manometric Techniques, Burgess, Minneapolis. 12. Sigler, K., Kotyk, A., Knotkova, A., and Opekarova, M. (198 I) Biochim. Biophys. Acta 643, 583-592.
13. Kotyk, A. (1967) Folia Microbial. 12, 121-131. 14. Kotyk, A., and HaSkovec, C. (1968) Fofia Microbiol.
13, 12-19.
15. Sigler, K., Knotkovl, Biochim.
Biophys.
A., and Kotyk, A. (1981) Acta 643,
572-582.
16. Jimenez, M. A., Sigler, K., Herrera, L. S., and Kotyk, A. (1980) Biolbgia (Bratislava) 35, 167171.
BIOCHEMISTRY
123, 201-204
(1982)
A Simple Spectrophotometric Method for Determining Proton Release from Yeast Cells CARLOSPASCUALAND Centro
National
ARNO~T KOTYK
de Investigaciones CientQicas. La Habana, Cuba, Czechoslovak Academy of Sciences, 142 20 Prague,
and Institute Czechoslovakia
of Microbiology,
Received December 28, 1981 A simple spectrophotometric acid-base indicator method was developed to determine the rate of proton extrusion after addition of a fermentable hexose to a yeast cell suspension. Unlike the conventional pH-electrode measurements the method gives linear response to changes in proton concentration and makes rapid kinetic determinations possible.
Increasing attention has been focused on the electrochemical potential gradient of protons across cell membranes (1). The proton gradient appears to be the source of energy for ATP synthesis, as well as for active transport in cell organelles (2,3), bacteria (4), and yeast (5) and algal (6) plasma membranes. Determination of the magnitude and mechanism of formation of the transmembrane pH gradient has become a major task in the bioenergetics of lower eukaryotes. Under whatever conditions a change in pH takes place it will depend on the relative buffering capacities of the internal and external media. Changes in intracellular pH are usually monitored with a pH-sensitive glass electrode (5,7,8). However, the time course of changes of actual concentration of H+ ions is less evident since it must be deduced from the logarithmic record. Likewise, small changes in H+ concentration may be difficult to assess. In the present work, a simple indicator method has been used to overcome these shortcomings of the now standard pH estimations. MATERIALS
spectrophotometrically the rate of acid produced when yeast cells are incubated with a fermentable hexose. Using the acidic peak of the spectrum at 540 nm (see Fig. 1) we obtained a linear relationship between addition of protons to methyl red solution and absorbance. For reactions where proton liberation occurs at pH not very far from the pK of the indicator (5.0 for methyl red) the change in absorbance of the dye becomes a quantitative measure of proton concentration. (It was checked that the approximately 0.6 mM methyl red solution had no adverse effect on cell fermentation or respiration, as well as on intracellular pH; for the method see Ref (9).) Strain and culture conditions. The strain 196-2 (a-his-6) of Saccharomyces cerevisiae was used for the study. Cells were grown on minimal medium containing 2% glucose as described previously (10) and were harvested at the end of the exponential phase. The cells were washed with deionized water at 4’C. Preparation of indicator solution. The indicator solution was prepared by dissolving 100 mg methyl red in 20 ml of 20 mM NaOH and diluting to 500 ml with deionized water. It is important that any excess of NaOH in the indicator solution be neutralized with HCl. The amount of acid added for neu-
AND METHODS
Principle of the method. It is based on the use of methyl red as an indicator to measure 201
0003-2697/82/090201-04$02.00/0 Copyright 0 1982 by Academic Press, Inc. All rights of reproduction in any form reserved.
202
PASCUAL
AND KOTYK
WAVELENGTH
( nm 1
I. Absorption spectrum of the acidic and basic forms of methyl red. Methyl red solution (27 0.2 M HCI (acidic form) and in 0.2 M NaOH (basic form) was placed in a cuvette of I-cm light path. The absorption spectrum was registered at a scanning speed of 4 nm s-’ and a recording velocity of 0.2 cm s-‘. FIG. PM) in
tralization is determined by placing a certain volume of the indicator solution in a spectrophotometric cuvette and adding 20-~1 portions of 0.1 M HCl until a change of ab-
sorbance at 540 nm occurs. The amount of HCl added just before this change takes place is used for calculating the amount needed for neutralizing excess NaOH in the 500-ml stock solution. A calibration curve is then used to determine the value of absorbance which corresponds to a given quantity of protons. Usually a change of absorbance of 0.055 at 540 nm corresponds to 10 nmol of protons released in the reaction. Measurement of proton concentration changes. Yeast cell suspension (50 ~1,at 40-
I 20
nmol
H
2. Changes in indicator absorbance due to proton liberation from yeast cells after addition of different sugars. Fifty microliters of a yeast suspension containing 42.7 mg fresh weight per milliliter was added to 2.5 ml of the indicator solution. At the times indicated by the arrows 4 pmol of the sugar was added. The velocity was recorded at a chart speed of 1 cm s-’ with scale expansion of 2.0 absorbance units. FIG.
80 mg/ml) is added to a l-cm cuvette containing 2.5 ml of methyl red solution. After equilibration, 50 ~1 of a specified sugar concentration is added and changes of absorbance are recorded at 540 nm at 30°C in a spectrophotometer (Pye-Unicam SP 1800 was used here). Glucose fermentation was measured by conventional manometric techniques (e.g., Ref. (11)) under the same conditions as the measurements of proton concentration. RESULTS
Figure 2 shows a typical record of absorbance obtained on adding various sugars to
DETERMINATION
OF PROTON
S. cerevisiae. No reaction can be detected if either substrate or cells are excluded from the reaction mixture. The rate of proton release as a function of cell density is linear up to about 6 mg cells in the reaction mixture (Fig. 3). Using higher cell concentrations for the measurement may result in underestimates, apparently due to the generation of buffering capacity by cells in the external medium (12). Proton release rates at different concentrations of glucose follow typical MichaelisMenten kinetics, with highly satisfactory linearity (correlation coefficient r = O-99), yielding a K,,, of 6.4 mM and I/ of 82 pmol min-’ (g yeast)-‘. Manometric measurements of fermentation rates at different concentrations of glucose using yeast cells under the same conditions yield rather similar parameters: Km = 4.8 mM, I/ = 62 pmol min-’ (g yeast)-‘. Addition of mannose which is metabolized more slowly than glucose elicits a 60% response in proton release while addition of nonmetabolizable sugars, such as D-xylose, D-arabinose (13), and D-galactose, prior to induction ( 14) causes no proton release. Addition of carbonylcyanide m-chlorophenylhydrazone as a proton conductor to shortcircuit the pH difference across the cell membrane had an immediate inhibitory effect on the proton release. DISCUSSION
Spectrophotometric measurement of the velocity of proton liberation by means of an indicator has several advantages as the reaction rate is constant and therefore direct calculations can be made. Moreover, the method is suitable for rapid kinetic measurements since it obviates the use of a pH electrode which generally has a slower response than a spectrophotometer. The detection of proton amounts as low as 10 nmol in 2.5 ml of suspension makes the method quite sensitive. The rate of proton liberation in the presence of 1.6 mM glucose observed here was
RELEASE
FROM
203
CELLS
009E +' 006. I
1
0
25
I
5.0 mg cells
I
75
FOG. 3. Rate of proton extrusion as function of cell concentration. Different amounts of yeast cells from a 77 mg/ml suspension were added to 2.5 ml of methyl red solution. The reaction was started after the addition of 2 pmol glucose. The velocity was registered as indicated in Fig. 2.
23 pmol min-’ (g yeast)-‘. Serrano (5) reported 15 to 30 pmol min-’ (g yeast))’ by measuring pH in yeast suspension in the presence of 25 mM glucose which, in extrapolation, would yield approximately 60 pmol min-’ (g yeast))’ by our method. This difference may be considered to be well within the spread of experimental values found with different batches of yeast. Proton extrusion appears to be linked to glycolysis even if there is no single proton pump responsible for this process (15). In fact, production of C02, release of some organic acids (succinic, malic, lactic), K+-H+ exchange, as well as an ATPase-type proton pump appear to be involved. Still, it is easily seen that nonfermentable sugars cause no proton extrusion from yeast cells. Likewise, JimCnez et al. (16) reported that glycolytic mutants of yeast even in the presence of metabolizable sugars are unable to acidify the medium due to the enzymatic defect, Further evidence to support this link lies in the fact that the K,,, value for glucose which causes proton extrusion is very similar to the K,,, for glucose fermentation. The proposed method could be used not only to study various kinetic parameters of the proton liberation process in yeast but also to quantify in a rapid and simple manner the degree of sugar fermentation.
PASCUAL
204 REFERENCES
1. Fillingame, R. H. (1980) Annu. Rev. Biochem. 49, 1079-1113. 2. Mitchell, P. (1961) Nature (London) 191, 144-148. 3. Whatley, F. R. (1975) in Energy Transformation in Biological Systems (Ciba Foundation Symposium), Vol. 31, pp. 41-61, Associated Scientitic Pub., Amsterdam. 4. Oesterhelt, D. (1975) in Energy Transformation in Biological Systems (Ciba Foundation Symposium), Vol. 31, pp. 147-165, Associated Scientific Pub., Amsterdam. 5. Serrano, R. (1980) Eur. J. Biochem. 105,419-424. 6. Komor, E. (1973) FEBS Lett. 38, 16-18. 7. Sigler, K., Knotkova, A., and Kotyk, A. (1978) Folia
Microbial.
23, 409-422.
8. Mitchell, P., and Moyle, J. (1965) Nature 208, 147-151.
(London)
AND KOTYK 9. Slavik, J, (1982) FEBS Left. 140, 22-26. 10. Herrera, L. S., Pascual, C., and Alvarez, X. (1976) Mol.
Gen. Genet.
144, 223-230.
1I. Umbreit, W. W., Burris, R. H., and Stauffer, J. F. (1957) Manometric Techniques, Burgess, Minneapolis. 12. Sigler, K., Kotyk, A., Knotkova, A., and Opekarova, M. (198 I) Biochim. Biophys. Acta 643, 583-592.
13. Kotyk, A. (1967) Folia Microbial. 12, 121-131. 14. Kotyk, A., and HaSkovec, C. (1968) Fofia Microbiol.
13, 12-19.
15. Sigler, K., Knotkovl, Biochim.
Biophys.
A., and Kotyk, A. (1981) Acta 643,
572-582.
16. Jimenez, M. A., Sigler, K., Herrera, L. S., and Kotyk, A. (1980) Biolbgia (Bratislava) 35, 167171.