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Sensitized chemiluminescence of coumarins as a possible component of plants ultraweak luminescence
262
Journal of Luminescence 40&41 (1988) 262 263 North-Holland, Amsterdam
SENSITIZED CHEMILUMINESCENCE OF COUMARINS AS A POSSIBLE COMPONENT OF PLANTS ULTRAWEAK LUMINESCENCE
Danuta SLAWINSKA and Janusz SLAWINSKI~ ~epartment of Physics, Agricultural University, Wojska Poiskiego 38/112 60-637 Poznan Institute of Physics, Pedagogical University, Podchorazych 2 30_O814 Krakow, Poland 2~3+)with the Cherniluminescence the peroxidative system: peroxide + peroxidase (Fe by 2—3 orders maximum intensity of 100-10000 photons/s.cm3 and hydrogen quantum yield 1012_io9 is enhanced of magnitude in the presence of coumarin derivatives. The emission spectra correspond to the fluorescence spectra of the derivative used.It is postulated that this sensitized chemiluminescence contributes to ultraweak luminescence of plants in the spectral region 380-500 nm.
INTRODUCTION Strongly fluorescing derivatives of coumarins
surement was repeated 3-5 times.Spectral distribution of chemiluminescence was measured combi-
e.g.7—hydroxycoumarin(umbell iferone) occur in
fling the single photon counting method with the
high concentrations in plant families Umbel life—
set of cut—off or interference filters. Details
rae and Rutaceae which found medical applica-
are given elsewhere2. Fluorescence excitation
tions. Umbell iferone is the precursor of photoa—
and emission spectra were recorded with a Perkin
ctive furocoumarins which are known to exert
Elmer MPF-LILIa spectrofluorimeter in the ratio
various biological actions, such as inactivation
mode. All spectra are corrected. Absorption
of DNA viruses, erythemia, mutagenesis etc under
spectra were measured using a Zeiss Specord UV—
irradiation with near UV. Hydroxycoumarins parti-
VIS apparatus and a 1 cm—path quartz cells.
R
cipate in many biological processes. Protection
6
3 1 2 0
R
invasion involves enzymatic hydroxylation and peroxidation of coumarins and polyphenols
,‘~
1
of plants from the physical damage and pathogen
2
.
We have studied the generation of electronically
R
excited states of coumarins (1_LI) coupled to the
-one (2);
enzymatic and model peroxidation processes. We
R1=CH3
1=R2=H
coumarin (1); R1—R2=OH
R2—OH
R1=H
R2=OF-l
esculetin
scopoletin
0 umbellife-
(3
(14
show that this sensitized chemiluminescence may contribute to spontaneous endogeneous ultraweak
Coumarin was from Aldrich, umbell iferone from
plants’ luminescence (metabolic chemilumines-
Merck-Schuchard, escul in and scopoletin from
cence).
K and K Labs. Other reagents were from
POCh
Gliwice, Poland. All solutions were always freshly prepared.
METHODS AND MATERIALS Chemiluminescence intensity vs the reaction time was measured using an RCA 5996A photomultipl ier tube connected to a K—20l
Zeiss integra-
ting recorder. Solutions were rapidly injected to a 10 ml quartz cuvette mounted on the front of the phototube. chemi— luminescence peak The hightreproducibil was 15-20/ ity and of each mea-
0022 2313/88/$03.50 © Elsevier Science Publishers By. (North-Holland Physics Publishing Division)
RESULTS The peroxidative system generating primary active oxygen species 0~and electronically excited species P~ 2~3~ + HRP (or Fe
H 2 02
~O
+..
x
.~
P~—
D. S/awinska, J Slawinski
~ P
+
/
Sensitized chemiluminescence of coumarins
photons (h V)
where HRP is horseradish peroxidase. emits weak chemiluminescence with the maximum intensity I m 3 and the quantum yield 102_lOLI photons/s.cm 0 = l0’~~—109in the spectral range 380—650 nm
263
hydrogen peroxide (pK a = 12.11,Fig. 2). In the investigated systems coumarins undergo slow lO~e) h reactions with 0 ( O OH., w ich lead x 2’ 2
4
and 1.3-1.6 um due to the mechanisms described previously3. In the presence of coumarins 1-14,
2
values of while I m and the 0 increase 2—3 orders of magnitude emission byspectrum corresponds
Ig ‘m
to the fluorescence spectrum of a coumarin den— vative being used (Fig.l
.
1
0~
These results mdi-
cate that sensitized chemiluminescence occurs
3579 1O
au. 0.5
~f2
,
I
20
~
1
~,
coumarins. All substrates of the above reactions are present in plant tissues and participate in their metabolism. Plant tissues are known to
I
28 32 3 cm1
“‘i0
FIGURE 1 Chemiluminescence, fluorescence excitation (1 and emission (2) spectra of 50 1jM umbelliferone with 60 mM hydrogen peroxide and 0.1 ~g/ml HRP at pH(2 6.5. of spectra (1) at 1152 excitation of at Emission 31114 nm. All are nm, corrected,
that involves the excitation energy transfer from the primary excited species p~to coumarin: +
(1-14)
——~
p
+
The to a effect gradualof decrease pH2. on10 the6 enzymatic (1) and umbelliferone; non—enzymatic (2 the mMof sys~em KI the Fein concenctration containing CN + 10 mM 1 mM H of 3 202
I
24
-
FIGURE 2
5
I’L___L~
—
16
;
/
~
:1. liii ~ll 0
pH
gt II ii I ~
(l-11)~ ——~ (1-4)
+
h ~
Cheniluminescence emission spectra of px (380—
emit ultraweak luminescence in the spectral regionthe380800 nm that ofis lipids belivedandto polyphenols. originate from peroxidation There is a positive correlation between the I m and the HRP activity and the concentration of coumarins. Therefore the sensitized chemiluminescence of coumarins may contribute to plants’ luminescence in the spectral region 380—500 nm.
REFERENCES 1. D.H. Murray,J. Mendez and A. Brown, The Natural Coumarins (John Wiley and Son, Chister, New york,
1982).
—650 nm) cover the region ofabsorption (fluores-
2. R.F. Vassilev, Chemiluminescence in Liquidphase reactions, in: Progress in Reaction Kinetics, Vol .11, ed.J. Weisberger,1967 p.3O5.
cence excitation)
3. D. Slawinska and J. Slawinski, Low level
spectra of coumarins (1—4)
(320-500 nm). The function
I m= f (pH) correla—
tes with the pH—dependence of HRP activity inonethe enzymatic system while in the non-enzymatic it corresponds to the dissociation profile of
lu-
minescence from biological objects, in: Chemi 495-53l. and Bioluminescence, ed.J.G.Burr (Marcel Dek— ker Inc.,New York,1985) pp. This work was performed within the research project CPBP 01.12.37.1 PAN, Wroclaw.
Journal of Luminescence 40&41 (1988) 262 263 North-Holland, Amsterdam
SENSITIZED CHEMILUMINESCENCE OF COUMARINS AS A POSSIBLE COMPONENT OF PLANTS ULTRAWEAK LUMINESCENCE
Danuta SLAWINSKA and Janusz SLAWINSKI~ ~epartment of Physics, Agricultural University, Wojska Poiskiego 38/112 60-637 Poznan Institute of Physics, Pedagogical University, Podchorazych 2 30_O814 Krakow, Poland 2~3+)with the Cherniluminescence the peroxidative system: peroxide + peroxidase (Fe by 2—3 orders maximum intensity of 100-10000 photons/s.cm3 and hydrogen quantum yield 1012_io9 is enhanced of magnitude in the presence of coumarin derivatives. The emission spectra correspond to the fluorescence spectra of the derivative used.It is postulated that this sensitized chemiluminescence contributes to ultraweak luminescence of plants in the spectral region 380-500 nm.
INTRODUCTION Strongly fluorescing derivatives of coumarins
surement was repeated 3-5 times.Spectral distribution of chemiluminescence was measured combi-
e.g.7—hydroxycoumarin(umbell iferone) occur in
fling the single photon counting method with the
high concentrations in plant families Umbel life—
set of cut—off or interference filters. Details
rae and Rutaceae which found medical applica-
are given elsewhere2. Fluorescence excitation
tions. Umbell iferone is the precursor of photoa—
and emission spectra were recorded with a Perkin
ctive furocoumarins which are known to exert
Elmer MPF-LILIa spectrofluorimeter in the ratio
various biological actions, such as inactivation
mode. All spectra are corrected. Absorption
of DNA viruses, erythemia, mutagenesis etc under
spectra were measured using a Zeiss Specord UV—
irradiation with near UV. Hydroxycoumarins parti-
VIS apparatus and a 1 cm—path quartz cells.
R
cipate in many biological processes. Protection
6
3 1 2 0
R
invasion involves enzymatic hydroxylation and peroxidation of coumarins and polyphenols
,‘~
1
of plants from the physical damage and pathogen
2
.
We have studied the generation of electronically
R
excited states of coumarins (1_LI) coupled to the
-one (2);
enzymatic and model peroxidation processes. We
R1=CH3
1=R2=H
coumarin (1); R1—R2=OH
R2—OH
R1=H
R2=OF-l
esculetin
scopoletin
0 umbellife-
(3
(14
show that this sensitized chemiluminescence may contribute to spontaneous endogeneous ultraweak
Coumarin was from Aldrich, umbell iferone from
plants’ luminescence (metabolic chemilumines-
Merck-Schuchard, escul in and scopoletin from
cence).
K and K Labs. Other reagents were from
POCh
Gliwice, Poland. All solutions were always freshly prepared.
METHODS AND MATERIALS Chemiluminescence intensity vs the reaction time was measured using an RCA 5996A photomultipl ier tube connected to a K—20l
Zeiss integra-
ting recorder. Solutions were rapidly injected to a 10 ml quartz cuvette mounted on the front of the phototube. chemi— luminescence peak The hightreproducibil was 15-20/ ity and of each mea-
0022 2313/88/$03.50 © Elsevier Science Publishers By. (North-Holland Physics Publishing Division)
RESULTS The peroxidative system generating primary active oxygen species 0~and electronically excited species P~ 2~3~ + HRP (or Fe
H 2 02
~O
+..
x
.~
P~—
D. S/awinska, J Slawinski
~ P
+
/
Sensitized chemiluminescence of coumarins
photons (h V)
where HRP is horseradish peroxidase. emits weak chemiluminescence with the maximum intensity I m 3 and the quantum yield 102_lOLI photons/s.cm 0 = l0’~~—109in the spectral range 380—650 nm
263
hydrogen peroxide (pK a = 12.11,Fig. 2). In the investigated systems coumarins undergo slow lO~e) h reactions with 0 ( O OH., w ich lead x 2’ 2
4
and 1.3-1.6 um due to the mechanisms described previously3. In the presence of coumarins 1-14,
2
values of while I m and the 0 increase 2—3 orders of magnitude emission byspectrum corresponds
Ig ‘m
to the fluorescence spectrum of a coumarin den— vative being used (Fig.l
.
1
0~
These results mdi-
cate that sensitized chemiluminescence occurs
3579 1O
au. 0.5
~f2
,
I
20
~
1
~,
coumarins. All substrates of the above reactions are present in plant tissues and participate in their metabolism. Plant tissues are known to
I
28 32 3 cm1
“‘i0
FIGURE 1 Chemiluminescence, fluorescence excitation (1 and emission (2) spectra of 50 1jM umbelliferone with 60 mM hydrogen peroxide and 0.1 ~g/ml HRP at pH(2 6.5. of spectra (1) at 1152 excitation of at Emission 31114 nm. All are nm, corrected,
that involves the excitation energy transfer from the primary excited species p~to coumarin: +
(1-14)
——~
p
+
The to a effect gradualof decrease pH2. on10 the6 enzymatic (1) and umbelliferone; non—enzymatic (2 the mMof sys~em KI the Fein concenctration containing CN + 10 mM 1 mM H of 3 202
I
24
-
FIGURE 2
5
I’L___L~
—
16
;
/
~
:1. liii ~ll 0
pH
gt II ii I ~
(l-11)~ ——~ (1-4)
+
h ~
Cheniluminescence emission spectra of px (380—
emit ultraweak luminescence in the spectral regionthe380800 nm that ofis lipids belivedandto polyphenols. originate from peroxidation There is a positive correlation between the I m and the HRP activity and the concentration of coumarins. Therefore the sensitized chemiluminescence of coumarins may contribute to plants’ luminescence in the spectral region 380—500 nm.
REFERENCES 1. D.H. Murray,J. Mendez and A. Brown, The Natural Coumarins (John Wiley and Son, Chister, New york,
1982).
—650 nm) cover the region ofabsorption (fluores-
2. R.F. Vassilev, Chemiluminescence in Liquidphase reactions, in: Progress in Reaction Kinetics, Vol .11, ed.J. Weisberger,1967 p.3O5.
cence excitation)
3. D. Slawinska and J. Slawinski, Low level
spectra of coumarins (1—4)
(320-500 nm). The function
I m= f (pH) correla—
tes with the pH—dependence of HRP activity inonethe enzymatic system while in the non-enzymatic it corresponds to the dissociation profile of
lu-
minescence from biological objects, in: Chemi 495-53l. and Bioluminescence, ed.J.G.Burr (Marcel Dek— ker Inc.,New York,1985) pp. This work was performed within the research project CPBP 01.12.37.1 PAN, Wroclaw.