- Email: [email protected]
Spectra of the formaldehyde-induced ultraweak luminescence from yeast cells
29
.I. Photochem. Photobiol. B: Biol., 21 (1993) 29-35
Spectra of the formaldehyde-induced yeast cells Marek Godlewski”, and Humio Inabac
Zenon
Rajfur”,
Janusz
ultraweak
Slawiiiski”,
Masaki
luminescence
Kobayashib,
from
Masashi
Usab
“Institute of Physics and Informatics, Pedagogical University, Podchorajch 2, 30-084 Krakow (Poland) bResearch Development Corporation of Japan, Biophoton Project, Sendai 980 (Japan) ‘Research Institute of Electrical Communication, Tohoku University, Katahira, Sendai 980 (Japan) (Received
October
13, 1992; accepted
May 6, 1993)
Abstract An increase in the intensity and distinct spectral changes of ultraweak luminescence from the yeast Saccharomyces cerevisiae were measured when the metabolism of cells was drastically altered. A small emission peak and a red emission band 680-850 nm appeared when air-dried cells were imbibed in water. Lethal concentrations of HCHO (O.Ol%-10%) elicited a 2500 fold increase of the emission intensity and distinct spectral alterations. A transient 500-580 nm emission appeared in the initial phase of interaction. Then a gradually increasing long-lasting red emission band centered around 620 nm predominated in the total spectral range covering 47O-S50 nm. These emissions were not correlated with minor changes in fluorescence emission and excitation spectra originating from tryptophan, flavins, and unidentified emitters.
Key words: Ultraweak
luminescence;
Yeast
cells; Formaldehyde;
1. Introduction Ultraweak luminescence (UL) emitted in the spectral range 200-900 nm at photon fluxes below cu. lo4 photons cmV2 s-’ has been detected from a wide variety of organisms and proved to be closely related to many important biological phenomena and vital functions [l, 21. Recently, a stress-enhanced UL from a variety of biosystems has been measured and claimed to be relevant to the perturbation of homeostasis and the adaptation capacity of biosystems to detrimental environmental conditions [3, 41. However, emission spectra of this UL have been reliably measured only in a few cases [5-81 because of extremely low intensity and its rapid changes. The lack of spectral characteristics makes it impossible to understand mechanisms of electronic excitation, energy transfer and emission associated with biological processes and structures. Numerous investigations on UL of yeast - a well defined eukaryotic type cell - have only determined kinetics and spectra from the exponential and stationary, normally growing (unperturbed) ceils [9]. Previous studies revealed dramatic
loll-1344/93/$6.00
Spectral
analysis;
Fluorescence
changes of UL during the transition of spores and seeds from a resting stage to an active metabolism [6, 10, 111, as well as during lethal interactions of cells with detrimental factors, e.g. poisons [2, 4, 5, 81. Preliminary evaluation of UL from the formaldehyde (HCHO)-stressed yeast cells has only determined approximate spectral range of UL and fluorescence [4, 12, 131. In this paper we report UL and fluorescence spectra from yeast cells Saccharomyces cerevzkiue in the resting (dried) stage, during their interaction with water (water-imbibed active cells) and in the lethal interaction induced by the toxic effect of formaldehyde (HCHO).
2. Experimental
details
2.1. Materials A 50 mg sample of Saccharomyces cerevkiae Hansen CBS 5926 (or S. boulurdii) in a lyophilized form containing lo9 living cells +6.5 mg lactose lH20 +93.5 mg saccharase purchased as a PerenteroF medicament (Thiemann Arzneimittel GmbH, Muster) was uniformly distributed on the
0 1993 - Elsevier Sequoia.
All rights reserved
M. Godlewski et al. / Spectra of the formaldehyde-induced
30
flat bottom of a glass or quartz tube. Sporadically, “Harvest Gold” bakery (GB) or Polish standard bakery yeast were used to compare results. In such a case 100 mg of air-dry yeast in 100 ml water or phosphate buffer pH 7.0 was incubated during 30 min at 25 “C and then centrifuged three times to obtain a pellet of pure cells. HCHO was from the Wako Pure Chemical Industries, Ltd., other chemicals from POCh Gliwice of analytical grade, water bidistilled. 2.2. Methods 2.2. I. Spectral distribution luminescence
of ultraweak
Spectral distribution of Ul was measured with two filter-equipped single photon counting-type spectrometers. The first contained 13 cut-off filters in the rotary disc placed on a R1333 Hamamatsu Photonics photomultiplier tube (PMT) sensitive in the spectral range 300-900 nm (A,, = 440 nm). The second was equipped with 34 cut-off filters and a UV-sensitive R 375 Hamamatsu Photonics PMT (180-650 nm). The filter discs were rotated stepwise by a microprocessor-controlled stepping motor, the total count and background also being measured during each rotation of the disc. Using a microcomputer, the spectral distribution of UL was calculated and corrected for the total intensity changes with time (I=f(t)), transmittance of filters and spectral sensitivity of the PMT, as described elsewhere [14, 151. Four replicate cultures of S. cerevfiiae Hansen and five samples of S. cerevisiae Harvest Gold were used. Each air-dried sample was inserted into a cuvette within a light-tight camera and allowed to reach a stable temperature and extinguish any possible delayed photoluminescence. Then the counting procedure was performed taking the maximum number of scans (cycles of the filter disc rotation). Next, 2.5 or 5 ml of water was injected into the cuvette and air bubbled through the yeast suspension to start the imbibition process. A rapid increase of UL followed by an immediate decay resulted in only 6-20 scans. In the third phase of each experiment, 0.24 or 0.48 ml of 37% HCHO was injected into the cuvette with the yeast culture which resulted in a gradual, but strong increase of UL. A high signalto-noise (S:N) ratio and slow dynamics of the emission allowed the accumulation of at least hundreds of scans. All operations were performed in a dim red light. To determine the vitality of yeast cells, the standard eosin-staining method was used.
ultraweak luminescence from yeast cells
2.2.2. Fluorescence
emission and excitation spectra
Fluorescence spectra were measured with a Perkin-Elmer MPF 44a spectrofluorimeter calibrated for rhodamine B6 in the spectral range 220-620 nm. 3. Results
and discussion
3.1. Ultraweak luminescence
spectra
The time course of the total intensity It=f(t) and the spectral distributionZ=f(A) for three stages of the experiment are shown in Figs. l-6. Air-dried resting yeast cells emit a quasi-stationary photon flux at the total rate (proportional to the intensity It) of about 40 counts per second (cps) at the S:N ratio 2.5 (Fig. l(A)). The emission spectrum covers a broad spectral range 450-860 nm with the statistically significant maximum at about 530 nm and a broad diffused red band centered around 640-670 nm (Fig. l(B)). The reproducibility of spectral measurements for three additional air-dried samples of S. cerevisiae Hansen is illustrated in Fig. 2. Owing to the quasi-stationary state of the emission in this stage, a large number of scans (100-200) could be taken and the reproducibility was satisfactory. UL from dry resting spores of fungus Entomophthora virulenta [lo] and seeds of cereal plants [l, 61 has been reported and shown to be correlated with the vitality of
I
5
460
560
660
760
’
860
t.hr
960
J.h-4 Fig. 1. Kinetics (A) of the signal (S) and noise (N) photocount rate I, =f(t) from 10y air-dried yeast cells Saccharomyces cerevisiae Hansen at 25 “C recorded over 5 h (199 scans). Spectral distribution (B) Z=f(A) of ultraweak luminescence averaged over 118 initial scans (cycles) and 199 --scans (5 h). Sampling time At for each total count I, (S + N) and background (N) was 5 s. The width of rectangles is equal to the difference between shortwave limits of the two consecutive filters. Error bars represent 50% confidence intervals. All spectra are corrected for the transmittance of filters, spectral sensitivity of the PMT and changes of the total emission Z, with time t.
M. Godlewski et al. / Spectra of the formaldehyde-induced
ultraweak luminescence from yeast cells
31
A
h hml Fig. 3. Kinetics (A) of the photocount rate signal (S) and noise (N) from 10’ yeast cells + saccharose + fructose after mixing with 5 ml water. Spectral distribution (B) of the luminescence averaged from 6 initial scans (6 min) and 9 scans (13.5 min) - --. The suspension bubbled with air At=5 s.
400 m
h,“rn
Fig. 2. Spectral distribution of ultraweak luminescence from three additional replica (air-dried samples) of Saccharomyces cerevisiae Hansen (Perenterol): A, 96 scans; B, 118 scans, and C, 198 scans.
the cells. Processes underlying these extremely low level emissions probably include a very slow autoxidation of cell components, e.g. lipids. Until now however, the spectral distribution of such emissions could not be reliably measured. Addition of water to the air-dried yeast cells causes a rapid increase in the count rate up to about 350 cps (S:N =20). Then, the signal amplitude decays almost to the background level (N- 16 cps). During the first 13 min of imbibition, when the water-cell interaction initiates the activation of cell metabolism, a temporal decrease of the 540 nm emission band and a distinct increase of the 675-830 nm band is observed (Fig. 2). Later, the red emission diminishes slightly and a spectrum which resembles the primary one (initial scans, solid line in Fig. 3) is restored after about 5 h. Other yeast strains give similar, though less clearly displayed results. This tendency for spectral alterations was also observed in previous experiments [13]. In this stage of the experiment (water-yeast cells interaction) great fluctuations of Zt and Z=f( A) occur and low precision is obtained because of (i) a low S:N-value, (ii) a limited number of scans, and (iii) a high value of the ratio of the rate of spectral changes during the observed process to
the rate of the filter disc rotation. The 50% confidence interval error bars represent the variability of the quasi-spectral single photon counting technique from one culture (sample). In order to illustrate the regularity of spectral distribution from replicate cultures, spectra from three additional replicates are shown in Fig. 4. Despite large experimental errors, the general character of the spectral distribution and its alterations seems to be satisfactorily reproduced. The addition of HCHO - a strong poison that denatures proteins - causes a dramatic increase in UL intensity concomitant with fast dying of yeast cells. The maximum amplitude of the signal exceeds more than 2400 times that of dry yeast cells (S:N- 100). Initially, diminution of the red emission at A> 675 nm and a pronounced increase of the 520 - 580 nm green emission are observed. A well resolved and statistically significant maximum at 540 nm is seen (Fig. 5). After approximately 30 min, when all cells are dead, again there is a relative increase of the red emission and the final spectrum covers the 450-880 nm range with two slightly marked diffuse maxima at 570 and 620 nm. Owing to relatively high Zt and long-lasting emission, the precision of measurements is satisfactory. Other varieties of yeast give very similar results, as shown in Fig. 6. Spectral measurements with the UV-sensitive R375 PMT reveal a weak but statistically significant emission band centered at 380 nm which arose after the addition of 3% HCHO to the yeast cell suspension. These measurements confirm that there is no emission in the spectral range 390-450 nm from S. cerevisiae Hansen and Harvest Gold.
32
M. Godlewski et al. / Spectra of the formaldehyde-induced
-. Fig. 4. Spectral distribution of luminescence from three independent replicas of 5. cerevisiae Hansen treated with water and air. A, 13 min after injection of water, 9 scans; B, 15 min, 28 scans; C, 11 min, 21 scans.
400
500
600
700
800
900
A Cnml Fig. 5. Kinetics (A) and (C) Zt=f(t) of the signal (S) and noise (N) of the photocount rate from the active yeast cell suspension (as in Fig. 3) treated with 3% formaldehyde and aerated. The density of yeast cell is 2X lOa cells ml-‘. Spectral distribution (B) Z=f(A) averaged from 21 initials scans (31.5 min after addition of HCHO) and from 315 scans (7.8 h) -- --. Art=5 s.
ultraweak luminescence from yeast cells
Fig. 6. Spectral distribution of ultraweak luminescence from active cells of S. cerevisiae Harvest Gold treated with 3.5% HCHO measured over varying time of interaction. A, 10 min, 5 scans; B, 12-55 min, 30 scans; C, 57-270 min, 80 scans.
3.2. Fluorescence spectra A strong emission band with the A,,=333 nm, spectral bandwidth AA=56 &2 nm and A,,= 285-290 nm is the predominant component of the fluorescence from suspensions and supernatants (in this case about 40 times weaker) of yeast cells. This band (Fig. 7) is similar to the spectrum which is produced from the singlet state IL, of tryptophan (trp) in protein when the trp is buried in the hydrophobic environment. The emission maximum (333 nm) occurs at shorter wavelengths than that of the trp residues which are exposed to water (= 350 nm). The excitation spectrum of cell suspensions displays two maxima at 286 and 298 nm rather than at 280 nm, which is the absorption maximum of trp. The shape of the excitation spectrum can be qualitatively explained in terms of an inner filtering effect. The minimum in the arises from absorption by species curve I,, =f(&) with fluorescence quantum yields which are less than that of the trp residues (e.g. tyrosine). This is confirmed by the excitation spectra of supernatants which display a single maximum at A,,
\ ;\lA
M. Godlewski et al. / Spectra of the formaldehyde-induced
I
I-
Q.U.
I
‘\
I’ \
/ I
/"..i?\
a.u
\
\ \
j
!
ii
I
i i
/!
1k :
:
:
:
\ \ \ \ \
ultraweak luminescence from yeast cells
33
I
aL
i i i
y3irz-z \\ h,k-ml
:
\
\
\
\
350
\
\
‘.
400 Lo-m
Fig. 7. Fluorescence emission spectra of the yeast (Saccharomyces cerevbiae Hansen) recorded at the A,,=290 nm and cell density 2 x 10s. Intact cell suspension in water - - - - and in 3% formaldehyde after 1 h of interaction -. The insert shows excitation spectra (A,, = 333 nm) of a yeast cell suspension in 3% HCHO after 30 min of interaction -.-.-.and from supernatant -. All spectra are corrected.
285 nm and a clear red shift of the emission time (&lX =348 mu). After a long interaction between yeast cells and HCHO, only a very weak decrease of the emission band at 333 nm is observed (Fig. 7, solid line) without measurable changes in Amax, AA or its shape. Therefore, one may conclude that the polarity of the trp residue is not significantly changed during relatively short interaction time. This would be in agreement with the formation of hydrophobic CH,-bridges between protein chains in the reaction between the NH- and/or NH,-groups of the proteins and HCHO. Other components of yeast fluorescence are at least 10 times weaker than the trp emission/excitation. Among them, a clear though low intensity emission band at 528 nm (A,= 220, 362 and 440 nm) undoubtedly arises from the ‘S of flavins. No marked changes in its intensity are found for intact, HCHO- or thermally-inactivated yeast cells both in cell suspensions and supernatants. An HCHO-sensitive weak emission at 435-O nm (&= 360-375 nm) is observed in intact cell suspensions and supernatants (Fig. 8). This may originate from the fluorescence of thiamine derivatives, e.g. thiochrome (A,, = 425-435 nm, A -370 nm) which is the product of thiamine ozdation. N-formylkynurenin derivatives, which are products of the metabolic oxidative degradation reactions of trp, also emit in this region. The above assignments are compatible with the postulated
450
500 A,,[nml
Fig. 8. Fluorescence emission spectra (A, = 360 nm) from intact yeast cell suspension (0) and for varying time indicated by numbers, in min) of interaction with 3% HCHO. Insert shows excitation spectrum from the intact cell suspension recorded at A,,=440 nm.
radical oxidation reactions of HCHO in yeast, relevant to the promoting effect of HCHO on the UL of cells [12, 131. It has been suggested that the UL-promoting effect of HCHO may be a result of the formation of efficiently fluorescing Schiff base [16] which arises in the reaction between -NH2 and > C=O groups. However, we observed that solutions of HCHO incubated with aminoacids, amines, proteins, or nucleic acids under physiological conditions (20-30 “C, pH 4-8 multimolar concentrations of reagents) exhibit neither UL nor fluorescence. Therefore, the proposed [16] interpretation may be not valid unless certain enzymes and/or active cell components are not inactivated by the denaturating action of HCHO or catalytically active heavy metal ions are present in the reaction system.
4. Conclusions The above results prove that UL associated with metabolic processes elicited by the action of water or HCHO on yeast cells is distinctly altered in intensity and spectral distribution. Comparison of UL and fluorescence spectra leads to the conclusion that there is no correlation between them and the denaturation of yeast protein. Questions arise about the mechanisms of electronic excitation and photon emission processes in the studied system. These emissions can most
34
M. Godlewski et al. I Spectra of the formaldehyde-induced
probably be assigned to radical oxidation reactions and the decomposition of lipid peroxides in biomembranes which are known to generate excited carbonyls
kind permission to use a Perkin-Elmer spectrofluorimeter and for valuable discussions.
References 1 D. Slawinski and J. Slawinski, Biological chemiluminescence,
2 3
4
5
H,>C=O+H-O-O-R----+ H,>C-OH+R-O-O and subsequent radical chain reactions with membrane lipids and OZ. Such an interpretation accounts for the strong oxygen dependence of the HCHO-enhanced UL of yeast and other organisms [4, 7, 81. However, detailed mechanisms of chemiexcitation, possible energy transfer and emission require further investigation. Moreover, from the comparison of UL and fluorescence data it is evident that the dynamics and the range of intensity changes are greater for UL than for Auorescence. This evidence confirms a new methodological approach which proposes the use of UL parameters, particularly the analysis of stochastic processes such as single photocount time series, to evaluate, quantitatively, the perturbation degree and capacity of homeostasis [3, 41. This knowledge about spectral characteristics is also necessary for the correct calculation of real UL intensity changes elicited by stress factors.
6
7
8
9
10
11
12
Acknowledgments The research was supported by grants from the Biophoton Project, Research Development Corporation of Japan (JRDC) and (partially) the Ministry of National Education, Poland. The authors express their gratitude to Professors Anna and Jacek Koziol from the Institute of Commodity Science, Academy of Economics in Poznan for
ultraweak luminescence from yeast cells
13
14
Photochem. Photobiol., 37 (1983) 709-715. D. Slawinska and J. Slawinski, Low level luminescence from biological objects, in J.G. Burr (ed.), Chemi- and Bioluminescence, Marcel Dekker, New York, 1985. F.A. Popp (ed.), Biophoton emission, E@erientia, 44 (1988) 543-600. B. Kochel, Perturbed living organisms: a cybernetic approach founded on photon emission stochastic processes, Kybemetes, 19 (1990) 16-25. J. Slawinski, A. Ezzahir, M. Godlewski, B. Kochel, T. Kwiecinska, Z. Rajfur, D. Sitko and D. Wierzuchowska, Stress-induced photon emission from perturbed organisms, Experientia, 48 (1992) 1041-1058. B. Ruth, Experimental investigation of ultraweak photon emission, in F.A. Popp, U. Warnke, H.L. Koenig and W. Peschka (eds.), Electromagnetic Bioinfonnation, 2nd edn., Urban & Schwarzenberg, Miinchen, 1989, pp. 128143. J. Slawinski, E. Grabikowski and L. Ciesla, Spectral distribution of ultraweak luminescence from germinating plants,J. Lumin., 24125 (1981) 791-794. D. Slawinski and K. Polewski, Spectral analysis of plant chemiluminescence: participation of polyphenols and aldehydes in light-producing reactions, in B. Jezowska-Trzebiatowska, B. Kochel, J. Slawinski and W. Strek (eds.), Photon Emission from Biological Systems, World Scientific, Singapore, 1987, pp. 226-247. H. Watanabe, M. Kobayashi, S. Suzuki, M. Usa and H. Inaba, Aldehyde-enhanced photon emission from crude extracts of soybean seedlings, in P. Stanley and L.J. Kricka (eds.), Bioluminescence and Chemiluminescence, John Wiley, London, 1991, pp. 273-276. T.I. Quickenden, M.J. Comarmond and R.N. Tilbury, Ultraweak bioluminescence spectra of stationary phase Saccharomyces cerevisiae and Schizosaccharomyces pombe, Photothem. Photobiol., 41 (1985) 611-615. J. Slawinski, I. Majchrowicz and E. Grabikowski, Ultraweak luminescence from germinating resting spores of Entomophtora virulenta, Acta Mycol., 17 (1981) 127-135. R. Saeki, M. Kobayashi, M. Usa, B. Yoda, T. Miyazawa and H. Inaba, Low-level chemiluminescence of waterimbided soybean seeds, Agr. Biol. Chem., 53 (1989) 33113312. M. Godlewski, M. Krol, Z. Rajfur and D. Sitko, Influence of environmental conditions on the luminescence from Saccharomyces cerevisiae, in B. Jezowska-Trzebiatowska, B. Kochel, J. Slawinski and W. Strek (eds.), Biological Luminescence, World Scientific, Singapore, 1990, pp. 182196. A. Ezzahir, M. Godlewski, M. Krol, T. Kwiecinska, Z. Rajfur, D. Sitko and J. Slawinski, The influence of environmental factors on the ultraweak luminescence from yeast Saccharomyces cerevisiae, Bioelectrochem. Bioenergetics, 27 (1992) 57-61. H. Inaba, Super-high sensitivity system for detection and spectral analysis of ultraweak photon emission from
M. Godlewski et al. / Spectra of the formaldehyde-induced biological ceils and tissues, Erperientia, 44 (1988) 550559. 15 M. Kobayashi, M. Usa, S. Agatsuma, S. Suzuki, H. Watanabe, Y. Taguchi, H. Sekino and H. Inaba, Development and application of highly sensitive systems for ultraweak biophoton
ultraweak luminescence from yeast cells
35
measurement and analysis-progress in biophoton research, Photomed. Photobiol., 12 (1990) 87-100. 16 N.V. Levina, S.N. Orlov, Yu.M. Petrusevich and B.N. Tarusov, Formaldehyde-induced chemiluminescence in model systems protein-lipid, Biophysics, 21 (1976) 420-423.
.I. Photochem. Photobiol. B: Biol., 21 (1993) 29-35
Spectra of the formaldehyde-induced yeast cells Marek Godlewski”, and Humio Inabac
Zenon
Rajfur”,
Janusz
ultraweak
Slawiiiski”,
Masaki
luminescence
Kobayashib,
from
Masashi
Usab
“Institute of Physics and Informatics, Pedagogical University, Podchorajch 2, 30-084 Krakow (Poland) bResearch Development Corporation of Japan, Biophoton Project, Sendai 980 (Japan) ‘Research Institute of Electrical Communication, Tohoku University, Katahira, Sendai 980 (Japan) (Received
October
13, 1992; accepted
May 6, 1993)
Abstract An increase in the intensity and distinct spectral changes of ultraweak luminescence from the yeast Saccharomyces cerevisiae were measured when the metabolism of cells was drastically altered. A small emission peak and a red emission band 680-850 nm appeared when air-dried cells were imbibed in water. Lethal concentrations of HCHO (O.Ol%-10%) elicited a 2500 fold increase of the emission intensity and distinct spectral alterations. A transient 500-580 nm emission appeared in the initial phase of interaction. Then a gradually increasing long-lasting red emission band centered around 620 nm predominated in the total spectral range covering 47O-S50 nm. These emissions were not correlated with minor changes in fluorescence emission and excitation spectra originating from tryptophan, flavins, and unidentified emitters.
Key words: Ultraweak
luminescence;
Yeast
cells; Formaldehyde;
1. Introduction Ultraweak luminescence (UL) emitted in the spectral range 200-900 nm at photon fluxes below cu. lo4 photons cmV2 s-’ has been detected from a wide variety of organisms and proved to be closely related to many important biological phenomena and vital functions [l, 21. Recently, a stress-enhanced UL from a variety of biosystems has been measured and claimed to be relevant to the perturbation of homeostasis and the adaptation capacity of biosystems to detrimental environmental conditions [3, 41. However, emission spectra of this UL have been reliably measured only in a few cases [5-81 because of extremely low intensity and its rapid changes. The lack of spectral characteristics makes it impossible to understand mechanisms of electronic excitation, energy transfer and emission associated with biological processes and structures. Numerous investigations on UL of yeast - a well defined eukaryotic type cell - have only determined kinetics and spectra from the exponential and stationary, normally growing (unperturbed) ceils [9]. Previous studies revealed dramatic
loll-1344/93/$6.00
Spectral
analysis;
Fluorescence
changes of UL during the transition of spores and seeds from a resting stage to an active metabolism [6, 10, 111, as well as during lethal interactions of cells with detrimental factors, e.g. poisons [2, 4, 5, 81. Preliminary evaluation of UL from the formaldehyde (HCHO)-stressed yeast cells has only determined approximate spectral range of UL and fluorescence [4, 12, 131. In this paper we report UL and fluorescence spectra from yeast cells Saccharomyces cerevzkiue in the resting (dried) stage, during their interaction with water (water-imbibed active cells) and in the lethal interaction induced by the toxic effect of formaldehyde (HCHO).
2. Experimental
details
2.1. Materials A 50 mg sample of Saccharomyces cerevkiae Hansen CBS 5926 (or S. boulurdii) in a lyophilized form containing lo9 living cells +6.5 mg lactose lH20 +93.5 mg saccharase purchased as a PerenteroF medicament (Thiemann Arzneimittel GmbH, Muster) was uniformly distributed on the
0 1993 - Elsevier Sequoia.
All rights reserved
M. Godlewski et al. / Spectra of the formaldehyde-induced
30
flat bottom of a glass or quartz tube. Sporadically, “Harvest Gold” bakery (GB) or Polish standard bakery yeast were used to compare results. In such a case 100 mg of air-dry yeast in 100 ml water or phosphate buffer pH 7.0 was incubated during 30 min at 25 “C and then centrifuged three times to obtain a pellet of pure cells. HCHO was from the Wako Pure Chemical Industries, Ltd., other chemicals from POCh Gliwice of analytical grade, water bidistilled. 2.2. Methods 2.2. I. Spectral distribution luminescence
of ultraweak
Spectral distribution of Ul was measured with two filter-equipped single photon counting-type spectrometers. The first contained 13 cut-off filters in the rotary disc placed on a R1333 Hamamatsu Photonics photomultiplier tube (PMT) sensitive in the spectral range 300-900 nm (A,, = 440 nm). The second was equipped with 34 cut-off filters and a UV-sensitive R 375 Hamamatsu Photonics PMT (180-650 nm). The filter discs were rotated stepwise by a microprocessor-controlled stepping motor, the total count and background also being measured during each rotation of the disc. Using a microcomputer, the spectral distribution of UL was calculated and corrected for the total intensity changes with time (I=f(t)), transmittance of filters and spectral sensitivity of the PMT, as described elsewhere [14, 151. Four replicate cultures of S. cerevfiiae Hansen and five samples of S. cerevisiae Harvest Gold were used. Each air-dried sample was inserted into a cuvette within a light-tight camera and allowed to reach a stable temperature and extinguish any possible delayed photoluminescence. Then the counting procedure was performed taking the maximum number of scans (cycles of the filter disc rotation). Next, 2.5 or 5 ml of water was injected into the cuvette and air bubbled through the yeast suspension to start the imbibition process. A rapid increase of UL followed by an immediate decay resulted in only 6-20 scans. In the third phase of each experiment, 0.24 or 0.48 ml of 37% HCHO was injected into the cuvette with the yeast culture which resulted in a gradual, but strong increase of UL. A high signalto-noise (S:N) ratio and slow dynamics of the emission allowed the accumulation of at least hundreds of scans. All operations were performed in a dim red light. To determine the vitality of yeast cells, the standard eosin-staining method was used.
ultraweak luminescence from yeast cells
2.2.2. Fluorescence
emission and excitation spectra
Fluorescence spectra were measured with a Perkin-Elmer MPF 44a spectrofluorimeter calibrated for rhodamine B6 in the spectral range 220-620 nm. 3. Results
and discussion
3.1. Ultraweak luminescence
spectra
The time course of the total intensity It=f(t) and the spectral distributionZ=f(A) for three stages of the experiment are shown in Figs. l-6. Air-dried resting yeast cells emit a quasi-stationary photon flux at the total rate (proportional to the intensity It) of about 40 counts per second (cps) at the S:N ratio 2.5 (Fig. l(A)). The emission spectrum covers a broad spectral range 450-860 nm with the statistically significant maximum at about 530 nm and a broad diffused red band centered around 640-670 nm (Fig. l(B)). The reproducibility of spectral measurements for three additional air-dried samples of S. cerevisiae Hansen is illustrated in Fig. 2. Owing to the quasi-stationary state of the emission in this stage, a large number of scans (100-200) could be taken and the reproducibility was satisfactory. UL from dry resting spores of fungus Entomophthora virulenta [lo] and seeds of cereal plants [l, 61 has been reported and shown to be correlated with the vitality of
I
5
460
560
660
760
’
860
t.hr
960
J.h-4 Fig. 1. Kinetics (A) of the signal (S) and noise (N) photocount rate I, =f(t) from 10y air-dried yeast cells Saccharomyces cerevisiae Hansen at 25 “C recorded over 5 h (199 scans). Spectral distribution (B) Z=f(A) of ultraweak luminescence averaged over 118 initial scans (cycles) and 199 --scans (5 h). Sampling time At for each total count I, (S + N) and background (N) was 5 s. The width of rectangles is equal to the difference between shortwave limits of the two consecutive filters. Error bars represent 50% confidence intervals. All spectra are corrected for the transmittance of filters, spectral sensitivity of the PMT and changes of the total emission Z, with time t.
M. Godlewski et al. / Spectra of the formaldehyde-induced
ultraweak luminescence from yeast cells
31
A
h hml Fig. 3. Kinetics (A) of the photocount rate signal (S) and noise (N) from 10’ yeast cells + saccharose + fructose after mixing with 5 ml water. Spectral distribution (B) of the luminescence averaged from 6 initial scans (6 min) and 9 scans (13.5 min) - --. The suspension bubbled with air At=5 s.
400 m
h,“rn
Fig. 2. Spectral distribution of ultraweak luminescence from three additional replica (air-dried samples) of Saccharomyces cerevisiae Hansen (Perenterol): A, 96 scans; B, 118 scans, and C, 198 scans.
the cells. Processes underlying these extremely low level emissions probably include a very slow autoxidation of cell components, e.g. lipids. Until now however, the spectral distribution of such emissions could not be reliably measured. Addition of water to the air-dried yeast cells causes a rapid increase in the count rate up to about 350 cps (S:N =20). Then, the signal amplitude decays almost to the background level (N- 16 cps). During the first 13 min of imbibition, when the water-cell interaction initiates the activation of cell metabolism, a temporal decrease of the 540 nm emission band and a distinct increase of the 675-830 nm band is observed (Fig. 2). Later, the red emission diminishes slightly and a spectrum which resembles the primary one (initial scans, solid line in Fig. 3) is restored after about 5 h. Other yeast strains give similar, though less clearly displayed results. This tendency for spectral alterations was also observed in previous experiments [13]. In this stage of the experiment (water-yeast cells interaction) great fluctuations of Zt and Z=f( A) occur and low precision is obtained because of (i) a low S:N-value, (ii) a limited number of scans, and (iii) a high value of the ratio of the rate of spectral changes during the observed process to
the rate of the filter disc rotation. The 50% confidence interval error bars represent the variability of the quasi-spectral single photon counting technique from one culture (sample). In order to illustrate the regularity of spectral distribution from replicate cultures, spectra from three additional replicates are shown in Fig. 4. Despite large experimental errors, the general character of the spectral distribution and its alterations seems to be satisfactorily reproduced. The addition of HCHO - a strong poison that denatures proteins - causes a dramatic increase in UL intensity concomitant with fast dying of yeast cells. The maximum amplitude of the signal exceeds more than 2400 times that of dry yeast cells (S:N- 100). Initially, diminution of the red emission at A> 675 nm and a pronounced increase of the 520 - 580 nm green emission are observed. A well resolved and statistically significant maximum at 540 nm is seen (Fig. 5). After approximately 30 min, when all cells are dead, again there is a relative increase of the red emission and the final spectrum covers the 450-880 nm range with two slightly marked diffuse maxima at 570 and 620 nm. Owing to relatively high Zt and long-lasting emission, the precision of measurements is satisfactory. Other varieties of yeast give very similar results, as shown in Fig. 6. Spectral measurements with the UV-sensitive R375 PMT reveal a weak but statistically significant emission band centered at 380 nm which arose after the addition of 3% HCHO to the yeast cell suspension. These measurements confirm that there is no emission in the spectral range 390-450 nm from S. cerevisiae Hansen and Harvest Gold.
32
M. Godlewski et al. / Spectra of the formaldehyde-induced
-. Fig. 4. Spectral distribution of luminescence from three independent replicas of 5. cerevisiae Hansen treated with water and air. A, 13 min after injection of water, 9 scans; B, 15 min, 28 scans; C, 11 min, 21 scans.
400
500
600
700
800
900
A Cnml Fig. 5. Kinetics (A) and (C) Zt=f(t) of the signal (S) and noise (N) of the photocount rate from the active yeast cell suspension (as in Fig. 3) treated with 3% formaldehyde and aerated. The density of yeast cell is 2X lOa cells ml-‘. Spectral distribution (B) Z=f(A) averaged from 21 initials scans (31.5 min after addition of HCHO) and from 315 scans (7.8 h) -- --. Art=5 s.
ultraweak luminescence from yeast cells
Fig. 6. Spectral distribution of ultraweak luminescence from active cells of S. cerevisiae Harvest Gold treated with 3.5% HCHO measured over varying time of interaction. A, 10 min, 5 scans; B, 12-55 min, 30 scans; C, 57-270 min, 80 scans.
3.2. Fluorescence spectra A strong emission band with the A,,=333 nm, spectral bandwidth AA=56 &2 nm and A,,= 285-290 nm is the predominant component of the fluorescence from suspensions and supernatants (in this case about 40 times weaker) of yeast cells. This band (Fig. 7) is similar to the spectrum which is produced from the singlet state IL, of tryptophan (trp) in protein when the trp is buried in the hydrophobic environment. The emission maximum (333 nm) occurs at shorter wavelengths than that of the trp residues which are exposed to water (= 350 nm). The excitation spectrum of cell suspensions displays two maxima at 286 and 298 nm rather than at 280 nm, which is the absorption maximum of trp. The shape of the excitation spectrum can be qualitatively explained in terms of an inner filtering effect. The minimum in the arises from absorption by species curve I,, =f(&) with fluorescence quantum yields which are less than that of the trp residues (e.g. tyrosine). This is confirmed by the excitation spectra of supernatants which display a single maximum at A,,
\ ;\lA
M. Godlewski et al. / Spectra of the formaldehyde-induced
I
I-
Q.U.
I
‘\
I’ \
/ I
/"..i?\
a.u
\
\ \
j
!
ii
I
i i
/!
1k :
:
:
:
\ \ \ \ \
ultraweak luminescence from yeast cells
33
I
aL
i i i
y3irz-z \\ h,k-ml
:
\
\
\
\
350
\
\
‘.
400 Lo-m
Fig. 7. Fluorescence emission spectra of the yeast (Saccharomyces cerevbiae Hansen) recorded at the A,,=290 nm and cell density 2 x 10s. Intact cell suspension in water - - - - and in 3% formaldehyde after 1 h of interaction -. The insert shows excitation spectra (A,, = 333 nm) of a yeast cell suspension in 3% HCHO after 30 min of interaction -.-.-.and from supernatant -. All spectra are corrected.
285 nm and a clear red shift of the emission time (&lX =348 mu). After a long interaction between yeast cells and HCHO, only a very weak decrease of the emission band at 333 nm is observed (Fig. 7, solid line) without measurable changes in Amax, AA or its shape. Therefore, one may conclude that the polarity of the trp residue is not significantly changed during relatively short interaction time. This would be in agreement with the formation of hydrophobic CH,-bridges between protein chains in the reaction between the NH- and/or NH,-groups of the proteins and HCHO. Other components of yeast fluorescence are at least 10 times weaker than the trp emission/excitation. Among them, a clear though low intensity emission band at 528 nm (A,= 220, 362 and 440 nm) undoubtedly arises from the ‘S of flavins. No marked changes in its intensity are found for intact, HCHO- or thermally-inactivated yeast cells both in cell suspensions and supernatants. An HCHO-sensitive weak emission at 435-O nm (&= 360-375 nm) is observed in intact cell suspensions and supernatants (Fig. 8). This may originate from the fluorescence of thiamine derivatives, e.g. thiochrome (A,, = 425-435 nm, A -370 nm) which is the product of thiamine ozdation. N-formylkynurenin derivatives, which are products of the metabolic oxidative degradation reactions of trp, also emit in this region. The above assignments are compatible with the postulated
450
500 A,,[nml
Fig. 8. Fluorescence emission spectra (A, = 360 nm) from intact yeast cell suspension (0) and for varying time indicated by numbers, in min) of interaction with 3% HCHO. Insert shows excitation spectrum from the intact cell suspension recorded at A,,=440 nm.
radical oxidation reactions of HCHO in yeast, relevant to the promoting effect of HCHO on the UL of cells [12, 131. It has been suggested that the UL-promoting effect of HCHO may be a result of the formation of efficiently fluorescing Schiff base [16] which arises in the reaction between -NH2 and > C=O groups. However, we observed that solutions of HCHO incubated with aminoacids, amines, proteins, or nucleic acids under physiological conditions (20-30 “C, pH 4-8 multimolar concentrations of reagents) exhibit neither UL nor fluorescence. Therefore, the proposed [16] interpretation may be not valid unless certain enzymes and/or active cell components are not inactivated by the denaturating action of HCHO or catalytically active heavy metal ions are present in the reaction system.
4. Conclusions The above results prove that UL associated with metabolic processes elicited by the action of water or HCHO on yeast cells is distinctly altered in intensity and spectral distribution. Comparison of UL and fluorescence spectra leads to the conclusion that there is no correlation between them and the denaturation of yeast protein. Questions arise about the mechanisms of electronic excitation and photon emission processes in the studied system. These emissions can most
34
M. Godlewski et al. I Spectra of the formaldehyde-induced
probably be assigned to radical oxidation reactions and the decomposition of lipid peroxides in biomembranes which are known to generate excited carbonyls
kind permission to use a Perkin-Elmer spectrofluorimeter and for valuable discussions.
References 1 D. Slawinski and J. Slawinski, Biological chemiluminescence,
2 3
4
5
H,>C=O+H-O-O-R----+ H,>C-OH+R-O-O and subsequent radical chain reactions with membrane lipids and OZ. Such an interpretation accounts for the strong oxygen dependence of the HCHO-enhanced UL of yeast and other organisms [4, 7, 81. However, detailed mechanisms of chemiexcitation, possible energy transfer and emission require further investigation. Moreover, from the comparison of UL and fluorescence data it is evident that the dynamics and the range of intensity changes are greater for UL than for Auorescence. This evidence confirms a new methodological approach which proposes the use of UL parameters, particularly the analysis of stochastic processes such as single photocount time series, to evaluate, quantitatively, the perturbation degree and capacity of homeostasis [3, 41. This knowledge about spectral characteristics is also necessary for the correct calculation of real UL intensity changes elicited by stress factors.
6
7
8
9
10
11
12
Acknowledgments The research was supported by grants from the Biophoton Project, Research Development Corporation of Japan (JRDC) and (partially) the Ministry of National Education, Poland. The authors express their gratitude to Professors Anna and Jacek Koziol from the Institute of Commodity Science, Academy of Economics in Poznan for
ultraweak luminescence from yeast cells
13
14
Photochem. Photobiol., 37 (1983) 709-715. D. Slawinska and J. Slawinski, Low level luminescence from biological objects, in J.G. Burr (ed.), Chemi- and Bioluminescence, Marcel Dekker, New York, 1985. F.A. Popp (ed.), Biophoton emission, E@erientia, 44 (1988) 543-600. B. Kochel, Perturbed living organisms: a cybernetic approach founded on photon emission stochastic processes, Kybemetes, 19 (1990) 16-25. J. Slawinski, A. Ezzahir, M. Godlewski, B. Kochel, T. Kwiecinska, Z. Rajfur, D. Sitko and D. Wierzuchowska, Stress-induced photon emission from perturbed organisms, Experientia, 48 (1992) 1041-1058. B. Ruth, Experimental investigation of ultraweak photon emission, in F.A. Popp, U. Warnke, H.L. Koenig and W. Peschka (eds.), Electromagnetic Bioinfonnation, 2nd edn., Urban & Schwarzenberg, Miinchen, 1989, pp. 128143. J. Slawinski, E. Grabikowski and L. Ciesla, Spectral distribution of ultraweak luminescence from germinating plants,J. Lumin., 24125 (1981) 791-794. D. Slawinski and K. Polewski, Spectral analysis of plant chemiluminescence: participation of polyphenols and aldehydes in light-producing reactions, in B. Jezowska-Trzebiatowska, B. Kochel, J. Slawinski and W. Strek (eds.), Photon Emission from Biological Systems, World Scientific, Singapore, 1987, pp. 226-247. H. Watanabe, M. Kobayashi, S. Suzuki, M. Usa and H. Inaba, Aldehyde-enhanced photon emission from crude extracts of soybean seedlings, in P. Stanley and L.J. Kricka (eds.), Bioluminescence and Chemiluminescence, John Wiley, London, 1991, pp. 273-276. T.I. Quickenden, M.J. Comarmond and R.N. Tilbury, Ultraweak bioluminescence spectra of stationary phase Saccharomyces cerevisiae and Schizosaccharomyces pombe, Photothem. Photobiol., 41 (1985) 611-615. J. Slawinski, I. Majchrowicz and E. Grabikowski, Ultraweak luminescence from germinating resting spores of Entomophtora virulenta, Acta Mycol., 17 (1981) 127-135. R. Saeki, M. Kobayashi, M. Usa, B. Yoda, T. Miyazawa and H. Inaba, Low-level chemiluminescence of waterimbided soybean seeds, Agr. Biol. Chem., 53 (1989) 33113312. M. Godlewski, M. Krol, Z. Rajfur and D. Sitko, Influence of environmental conditions on the luminescence from Saccharomyces cerevisiae, in B. Jezowska-Trzebiatowska, B. Kochel, J. Slawinski and W. Strek (eds.), Biological Luminescence, World Scientific, Singapore, 1990, pp. 182196. A. Ezzahir, M. Godlewski, M. Krol, T. Kwiecinska, Z. Rajfur, D. Sitko and J. Slawinski, The influence of environmental factors on the ultraweak luminescence from yeast Saccharomyces cerevisiae, Bioelectrochem. Bioenergetics, 27 (1992) 57-61. H. Inaba, Super-high sensitivity system for detection and spectral analysis of ultraweak photon emission from
M. Godlewski et al. / Spectra of the formaldehyde-induced biological ceils and tissues, Erperientia, 44 (1988) 550559. 15 M. Kobayashi, M. Usa, S. Agatsuma, S. Suzuki, H. Watanabe, Y. Taguchi, H. Sekino and H. Inaba, Development and application of highly sensitive systems for ultraweak biophoton
ultraweak luminescence from yeast cells
35
measurement and analysis-progress in biophoton research, Photomed. Photobiol., 12 (1990) 87-100. 16 N.V. Levina, S.N. Orlov, Yu.M. Petrusevich and B.N. Tarusov, Formaldehyde-induced chemiluminescence in model systems protein-lipid, Biophysics, 21 (1976) 420-423.