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The influence of environmental factors on the ultraweak luminescence from yeast Saccharomyces cerevisiae
57
Bioelectrochemistry and Bioenergetics, 27 (1992) 57-61
A section of .I Electroanal. Chem., and constituting Vol. 342 (1992) Elsevier Sequoia S.A., Lausanne
JEC BB 01460 Short communication
The influence of environmental factors on the ultraweak luminescence from yeast Saccharomyces cerevisiae l
Ali Ezzahir, Marek Godlewski, Malgorzata Krol, Teresa Kwiecinska, Zenon Rajfur, Dorota Sitko and Janusz Slawinski Institute of Physics, Pedagogical University ul. Podchorazych 2, 30-084 Krakow (Poland)
(Received 10 May 1991; in revised form 16 October 1991) INTRODUCTION
Ultraweak luminescence emitted from living organisms (O.l-lo4 photons s-l cm-*) is the manifestation of exergonic metabolic processes occurring mainly within biological membranes. This phenomenon depends on the energetics and rate of metabolic processes and the physiological state (homeostasis) of organism. The intensity I, kinetic pattern Z(t), spectral distribution Z(h) and other parameters of ultraweak luminescence may be influenced by physical, chemical and biological environmental factors [l-3]. Thus, changes of parameters of ultraweak luminescence elicited by external stimuli may be considered as a holistic response which provides information on the rate and character of exergonic oxidative processes. These processes lead to the formation of products in excited states. The results presented in this paper are an extension of our previous research [4-61 on ultraweak luminescence induced by chemical stress in yeast Saccharornyces cereuisiae. MATERIALS AND METHODS
In all measurements yeast cells Saccharomyces cereuisiae strains TF-29 and TF-32 in the late stationary phase (not synchronized culture) were used. Yeast cells were kept in the dark to avoid photo-delayed luminescence. Before each measurement yeast cells were incubated for 20 min in a solution containing 20 g/l of glucose and 8 g/l of NaCl. For each measurement, 2 ml of cell suspension (lo8 cells/ml) was injected into the measuring cuvette and (after 2 min) 2 ml of formaldehyde was added to obtain final concentrations as indicated in Fig. 2.
Presented at the Second International October 1989, Pleven, Bulgaria.
l
0302-4598/92/$05.00
School: Electromagnetic
Fields and Biomembranes,
0 1992 - Elsevier Sequoia S.A. All rights reserved
2-8
58
time/s Fig. 1. Kinetic curves of photon emission induced by 4% CH,O from live (1) and dead (2) yeast cells (lo8 cells cmm3); supernatant (3) obtained after centrifugation of heated cells (80°C 1 h) and pellet (4) of dead cells.
Chemiluminescence kinetics were measured using a photomultiplier tube FELL79 (sensitive in the region of 300-830 nm with maximum sensitivity at 430 nm) operating in the single photon counting mode. Simultaneously, oxygen consumption was monitored using a Clark electrode [5,6]. The luminescence induced by changes in the pH of the suspension medium was studied. All measurements were carried out at room temperature. RESULTS AND DISCUSSION
It is well established that oxygenated suspensions of yeast cell cultures spontaneously emit extremely low intensity electromagnetic radiation [71. In our experiments a stress-induced ultraweak luminescence elicited by the action of an environmental factor such as formaldehyde CH,O is studied. The results presented in Fig. 1 indicate that the intensity I, rise-time T and the integral intensity I, (which is proportional to the total number of emitted photons) strongly depend on the state of the yeast cells and are associated mainly with the vitality of cells. The analysis of Z,, and oxygen consumption dependence on formaldehyde concentration (Fig. 2), luminescence dependence on pH-value changes (Fig. 3) and kinetic curves Z = f(t) for various concentrations of formaldehyde shows that: (1) The I_ of ultraweak luminescence increases with an increase in the concentration of formaldehyde in the range 0.06254% by a factor of up to 70, while the initial rate of oxygen consumption (Fig. 2) decreases. Thus, it seems that ultraweak luminescence is not directly connected with the respiration of the cells but with the perturbation of their physiological state.
59
\dt$l/dt 5
[HCHO]/% Fig. 2. Number of counts in first 500 seconds (Ix) and the rate of 0, formaldehyde concentration.
uptake as the function of
(2) Decreasing the pH of the medium causes a decrease in the intensity of the ultraweak luminescence from yeast cells. Under basic conditions, the enhancement of ultraweak luminescence is probably due to the hydrolytic effect of hydroxyl ions (OH-) on the membrane. (3) The interaction between formaldehyde and components of the yeast cells is only partially enzymatic. Thermal denaturation of the cells reduces the intensity of ultraweak luminescence by only about 25% of the formaldehyde-induced lumines-
5-
4," % cn 3t a. 0 \ 0
2-
x
.
l-l
l-
o
6
.,.,.,.,.,.,.I', 7 8 9
Fig. 3. The dependence
10
11
12
13
14
PH of photon emission intensity on the pH of the yeast cell suspension.
60
Fig. 4. Schematic representation of CH,O S-sulphur, M-mannan, G-glucan.
action on the membrane
of yeast cells: P-phosphate,
cence from live cells. It is well known that CH,O denatures the functional and structural proteins in the cell membrane at the -CH,- cross-linkage with NHJNH groups as shown in Fig. 4. An inner membrane of yeast mitochondria contains formaldehyde dehydrogenase, CH,O : NAD+, oxidoreductase (EC. 1.2.1.1) - enzymes catalysing the NAD-dependent formation of S-formylglutathione [8] Glutathione_SH
+ CH,O + NAD+ Formaldehyde dehydrogenase Glutathione-SCOH
Glutathione-SCOH
+ NADH++ H+
+ H,O -S-formy’hydro’ase Glutathione-SH
+ HOOCH (formate)
For-mate, for which the inner mitochondrial membrane is permeable, is an effective oxidase inhibitor 19,101. It inhibits cytochrome c oxidase and NADH-cytochrome reductase activities [9] which brings about changes in the O,-uptake (see Fig. 2). A hypothetical mechanism of excited species generation, which at least partially explains experimental findings, may be proposed as follows: CH,O + H,O, + HOCH,OOH HOCH,OOH
+ Me”++ HOCH,O’+
where HOCH,OOH cu+‘) HOCH,O’+
0,
-
is a-hydroperoxide 2 HOCH,OO’
Me”+’ + OHand Me”+ are transient metal ions (Fe+2,
61
B
r----
HO-CO+O:O’+OC-H L-_---l ---____----
-I [H
$ -I
-
CH,O + HCOOH + H,O + 0,
--
OH]
L---____-____---A
The enthalpy of the last reaction is - AH = 545 kJ/mol. The important feature of this reaction is oxygen formation. Some fractions of the total amount of oxygen may be formed in the excited singlet states lAg and ‘Ci (for details see refs. 11 and 12). The visible region luminescence is attributed to excited carbonyl groups and excited singlet oxygen dimers formed during the decomposition of lipid peroxide. Since fatty acids with more than one double bond have not been found in bacteria and yeast cells, the present results show that the excited singlet oxygen cannot be formed-by lipid peroxide decomposition and cannot excite a secondary emission by reacting with polyunsaturated fatty acids. The observed ultraweak luminescence resembles in many respects (excluding the O,-dependence) biophysical radiation from brain cells reported by Reiber [13]. This radiation occurs only after an irreversible perturbation (e.g. ultrasonification, homogenization) of glial cells (oligodendrocytes) and is claimed to have a non-enzymatic origin. REFERENCES 1 A. Ezzahir, M. Godlewski, T. Kwiecinska, Z. Rajfur and K. Scieszka, in B. Jezowska-Trzebiatowska, B. Kochel, J. Slawinski and W. Strek, (Eds.), Photon Emission from Biological Systems, World Scientific Publishing Co., Singapore, 1989, p. 173. J. Slawinski, in B. Jezowska-Trzebiatowska, B. Kochel, J. Slawinski and W. Strek, (Eds.), Photon Emission from Biological Systems, World Scientific Publishing Co., Singapore, 1989, p. 49. B. Kochel, in B. Jezowska-Trzebiatowska, B. Kochel, J. Slawinski and W. Strek, (Eds.), Photon Emission from Biological Systems, World Scientific Publishing Co., Singapore, 1989, p. 101. A. Ezzahir, M. Godlewski, B. Kochel, T. Kwiecinska, Z. Rajfur, D. Sitko and J. Slawinski, Photon Emission-Based Characteristics of Biohomeostatis Perturbation, in H. Inaba and H. Wetaneba (Eds.), Advances in Biophotons, Springer-Verlag (in press). 5 A. Ezzahir, Z. Rajfur, P. Wolanska, K. Scieszka and J. Slawinski, Zagadn. Biofiz. Wsp&zesnej, 15 (1991128. 6 M. Godlewski, Z. Rajfur, A. Ezzahir, D. Sitko and M. Krol, in B. Jezowska-Trzebiatowska, B. Kochel, J. Slawinski and W. Strek, (Eds.), Photon Emission from Biological Systems, World Scientific Publishing Co., Singapore, 1989, p. 182. 7 T.I. Quickenden, M.J. Comarmond and R.N. Tilbury, Photochem Photobiol., 41 (1988) 611. 8 T. Scott and M. Eagleson, Concise Encyclopedia Biochemistry., Walter de Gruyter, Berlin, (1988). 9 J.B. Chappell and K.N. Haarchoff, in E.C. Slater, I. Kaniuga and L. Wojtczak, (Eds.), Biochemistry of Mitochondria, Academic Press, New York, 1967, p. 75. 10 P. Nicholls, Biochim. Biophys. Acta, 430 (1974) 13. 11 D. Slawinska and J. Slawinski, Anal. Chem., 47 (1975) 2101. 12 A. Ezzahir, T. Kwiecinska, J. Slawinski, M. Godlewski, D. Sitko, Z. Rajfur and D. Wierzuchowska, in B. Kochel and J.W. Dobrowolski, (Eds.), Integration for Common Future: Human Environment, World Scientific Publishing Co., Singapore, 1992 (in press). 13 H. Reiber, J. B&hem. Biolum., 4 (1989) 245.
Bioelectrochemistry and Bioenergetics, 27 (1992) 57-61
A section of .I Electroanal. Chem., and constituting Vol. 342 (1992) Elsevier Sequoia S.A., Lausanne
JEC BB 01460 Short communication
The influence of environmental factors on the ultraweak luminescence from yeast Saccharomyces cerevisiae l
Ali Ezzahir, Marek Godlewski, Malgorzata Krol, Teresa Kwiecinska, Zenon Rajfur, Dorota Sitko and Janusz Slawinski Institute of Physics, Pedagogical University ul. Podchorazych 2, 30-084 Krakow (Poland)
(Received 10 May 1991; in revised form 16 October 1991) INTRODUCTION
Ultraweak luminescence emitted from living organisms (O.l-lo4 photons s-l cm-*) is the manifestation of exergonic metabolic processes occurring mainly within biological membranes. This phenomenon depends on the energetics and rate of metabolic processes and the physiological state (homeostasis) of organism. The intensity I, kinetic pattern Z(t), spectral distribution Z(h) and other parameters of ultraweak luminescence may be influenced by physical, chemical and biological environmental factors [l-3]. Thus, changes of parameters of ultraweak luminescence elicited by external stimuli may be considered as a holistic response which provides information on the rate and character of exergonic oxidative processes. These processes lead to the formation of products in excited states. The results presented in this paper are an extension of our previous research [4-61 on ultraweak luminescence induced by chemical stress in yeast Saccharornyces cereuisiae. MATERIALS AND METHODS
In all measurements yeast cells Saccharomyces cereuisiae strains TF-29 and TF-32 in the late stationary phase (not synchronized culture) were used. Yeast cells were kept in the dark to avoid photo-delayed luminescence. Before each measurement yeast cells were incubated for 20 min in a solution containing 20 g/l of glucose and 8 g/l of NaCl. For each measurement, 2 ml of cell suspension (lo8 cells/ml) was injected into the measuring cuvette and (after 2 min) 2 ml of formaldehyde was added to obtain final concentrations as indicated in Fig. 2.
Presented at the Second International October 1989, Pleven, Bulgaria.
l
0302-4598/92/$05.00
School: Electromagnetic
Fields and Biomembranes,
0 1992 - Elsevier Sequoia S.A. All rights reserved
2-8
58
time/s Fig. 1. Kinetic curves of photon emission induced by 4% CH,O from live (1) and dead (2) yeast cells (lo8 cells cmm3); supernatant (3) obtained after centrifugation of heated cells (80°C 1 h) and pellet (4) of dead cells.
Chemiluminescence kinetics were measured using a photomultiplier tube FELL79 (sensitive in the region of 300-830 nm with maximum sensitivity at 430 nm) operating in the single photon counting mode. Simultaneously, oxygen consumption was monitored using a Clark electrode [5,6]. The luminescence induced by changes in the pH of the suspension medium was studied. All measurements were carried out at room temperature. RESULTS AND DISCUSSION
It is well established that oxygenated suspensions of yeast cell cultures spontaneously emit extremely low intensity electromagnetic radiation [71. In our experiments a stress-induced ultraweak luminescence elicited by the action of an environmental factor such as formaldehyde CH,O is studied. The results presented in Fig. 1 indicate that the intensity I, rise-time T and the integral intensity I, (which is proportional to the total number of emitted photons) strongly depend on the state of the yeast cells and are associated mainly with the vitality of cells. The analysis of Z,, and oxygen consumption dependence on formaldehyde concentration (Fig. 2), luminescence dependence on pH-value changes (Fig. 3) and kinetic curves Z = f(t) for various concentrations of formaldehyde shows that: (1) The I_ of ultraweak luminescence increases with an increase in the concentration of formaldehyde in the range 0.06254% by a factor of up to 70, while the initial rate of oxygen consumption (Fig. 2) decreases. Thus, it seems that ultraweak luminescence is not directly connected with the respiration of the cells but with the perturbation of their physiological state.
59
\dt$l/dt 5
[HCHO]/% Fig. 2. Number of counts in first 500 seconds (Ix) and the rate of 0, formaldehyde concentration.
uptake as the function of
(2) Decreasing the pH of the medium causes a decrease in the intensity of the ultraweak luminescence from yeast cells. Under basic conditions, the enhancement of ultraweak luminescence is probably due to the hydrolytic effect of hydroxyl ions (OH-) on the membrane. (3) The interaction between formaldehyde and components of the yeast cells is only partially enzymatic. Thermal denaturation of the cells reduces the intensity of ultraweak luminescence by only about 25% of the formaldehyde-induced lumines-
5-
4," % cn 3t a. 0 \ 0
2-
x
.
l-l
l-
o
6
.,.,.,.,.,.,.I', 7 8 9
Fig. 3. The dependence
10
11
12
13
14
PH of photon emission intensity on the pH of the yeast cell suspension.
60
Fig. 4. Schematic representation of CH,O S-sulphur, M-mannan, G-glucan.
action on the membrane
of yeast cells: P-phosphate,
cence from live cells. It is well known that CH,O denatures the functional and structural proteins in the cell membrane at the -CH,- cross-linkage with NHJNH groups as shown in Fig. 4. An inner membrane of yeast mitochondria contains formaldehyde dehydrogenase, CH,O : NAD+, oxidoreductase (EC. 1.2.1.1) - enzymes catalysing the NAD-dependent formation of S-formylglutathione [8] Glutathione_SH
+ CH,O + NAD+ Formaldehyde dehydrogenase Glutathione-SCOH
Glutathione-SCOH
+ NADH++ H+
+ H,O -S-formy’hydro’ase Glutathione-SH
+ HOOCH (formate)
For-mate, for which the inner mitochondrial membrane is permeable, is an effective oxidase inhibitor 19,101. It inhibits cytochrome c oxidase and NADH-cytochrome reductase activities [9] which brings about changes in the O,-uptake (see Fig. 2). A hypothetical mechanism of excited species generation, which at least partially explains experimental findings, may be proposed as follows: CH,O + H,O, + HOCH,OOH HOCH,OOH
+ Me”++ HOCH,O’+
where HOCH,OOH cu+‘) HOCH,O’+
0,
-
is a-hydroperoxide 2 HOCH,OO’
Me”+’ + OHand Me”+ are transient metal ions (Fe+2,
61
B
r----
HO-CO+O:O’+OC-H L-_---l ---____----
-I [H
$ -I
-
CH,O + HCOOH + H,O + 0,
--
OH]
L---____-____---A
The enthalpy of the last reaction is - AH = 545 kJ/mol. The important feature of this reaction is oxygen formation. Some fractions of the total amount of oxygen may be formed in the excited singlet states lAg and ‘Ci (for details see refs. 11 and 12). The visible region luminescence is attributed to excited carbonyl groups and excited singlet oxygen dimers formed during the decomposition of lipid peroxide. Since fatty acids with more than one double bond have not been found in bacteria and yeast cells, the present results show that the excited singlet oxygen cannot be formed-by lipid peroxide decomposition and cannot excite a secondary emission by reacting with polyunsaturated fatty acids. The observed ultraweak luminescence resembles in many respects (excluding the O,-dependence) biophysical radiation from brain cells reported by Reiber [13]. This radiation occurs only after an irreversible perturbation (e.g. ultrasonification, homogenization) of glial cells (oligodendrocytes) and is claimed to have a non-enzymatic origin. REFERENCES 1 A. Ezzahir, M. Godlewski, T. Kwiecinska, Z. Rajfur and K. Scieszka, in B. Jezowska-Trzebiatowska, B. Kochel, J. Slawinski and W. Strek, (Eds.), Photon Emission from Biological Systems, World Scientific Publishing Co., Singapore, 1989, p. 173. J. Slawinski, in B. Jezowska-Trzebiatowska, B. Kochel, J. Slawinski and W. Strek, (Eds.), Photon Emission from Biological Systems, World Scientific Publishing Co., Singapore, 1989, p. 49. B. Kochel, in B. Jezowska-Trzebiatowska, B. Kochel, J. Slawinski and W. Strek, (Eds.), Photon Emission from Biological Systems, World Scientific Publishing Co., Singapore, 1989, p. 101. A. Ezzahir, M. Godlewski, B. Kochel, T. Kwiecinska, Z. Rajfur, D. Sitko and J. Slawinski, Photon Emission-Based Characteristics of Biohomeostatis Perturbation, in H. Inaba and H. Wetaneba (Eds.), Advances in Biophotons, Springer-Verlag (in press). 5 A. Ezzahir, Z. Rajfur, P. Wolanska, K. Scieszka and J. Slawinski, Zagadn. Biofiz. Wsp&zesnej, 15 (1991128. 6 M. Godlewski, Z. Rajfur, A. Ezzahir, D. Sitko and M. Krol, in B. Jezowska-Trzebiatowska, B. Kochel, J. Slawinski and W. Strek, (Eds.), Photon Emission from Biological Systems, World Scientific Publishing Co., Singapore, 1989, p. 182. 7 T.I. Quickenden, M.J. Comarmond and R.N. Tilbury, Photochem Photobiol., 41 (1988) 611. 8 T. Scott and M. Eagleson, Concise Encyclopedia Biochemistry., Walter de Gruyter, Berlin, (1988). 9 J.B. Chappell and K.N. Haarchoff, in E.C. Slater, I. Kaniuga and L. Wojtczak, (Eds.), Biochemistry of Mitochondria, Academic Press, New York, 1967, p. 75. 10 P. Nicholls, Biochim. Biophys. Acta, 430 (1974) 13. 11 D. Slawinska and J. Slawinski, Anal. Chem., 47 (1975) 2101. 12 A. Ezzahir, T. Kwiecinska, J. Slawinski, M. Godlewski, D. Sitko, Z. Rajfur and D. Wierzuchowska, in B. Kochel and J.W. Dobrowolski, (Eds.), Integration for Common Future: Human Environment, World Scientific Publishing Co., Singapore, 1992 (in press). 13 H. Reiber, J. B&hem. Biolum., 4 (1989) 245.