- Email: [email protected]
The triplet state of pyridoxal
Vol. 32, No. 4, 1968
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
THE TRIPLET
STATE OF PYKIDOKAL*
John E. Wampler+
and Jorge
E. Churchich
Department of Biochemistry University of Tennessee Knoxville, Tennessee
Received
July 8, 1968
The absorption structures
of pyridoxal
laboratories
(Williams
and Martell,
1964).
known about
the
is
also
In spite
luminescence
detected
state
1954;
is
Metzler
and Snell,
of pyridoxal It
of pyridoxal
spectroscopy.
is
from the
triplet
1955;
at
Anderson little
low temperature
the purpose
is
in several
studies,
originates Evidence
of the
investigated
spectrophotometric
properties
transferred
distribution
have been
and phosphorescence.
by esr
energy
solution,
of these
the phosphorescence
excitation singlet
in aqueous
as the relative
and Neilands,
such as fluorescence show that
as well
spectra,
presented state
(77”
of this
in a triplet
is
note
to
state
that
K),
which
electronic
of pyridoxal
to the
of L-kynurenine.
Methods. Luminescence assembly
adopted
A rotating
shutter
phosphorescence recorded
for
(Aminco)
on a Tektronix
as a standard
work
for
in our
of the
samples.
532 oscilloscope
quantum
fellow
yield
conducted
The decay
was 0.1 second.
calculations
1966-69.
629
(Giltmore
Energy
Housing
(Churchich,
was desired
and the most
by the Atomic
in a Dewar
spectrofluorometer
was used when it
decay measurements
was supported
+ NDEA predoctoral
at 77’ K were
observations
properties
of phosphorescence
* This
measurements
to examine
1967). the
of phosphorescence probable
error
Benzophenone et al.,
Conmission,
grant
was in a group
was used
1955).
AT(40-l)-3553.
Vol. 32, No. 4, 1968
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
The esr measurementswere made with a Varian V 4502-12 spectrometer operating at 9.235 GHs. Illumination (Philips)
was provided by 500 watt high pressure mercury arc
and the wavelengths selected by a 500 mmBausch and Lombmonochromator.
Further details
on this apparatus are given by Ten Bosch z
al.,
1968. Low
temperature absorption spectra were obtained on a Perkin-Elmer Model 450 spectrophotometer.
Table I Luminescence characteristics of pyridoxal in the mixed solvent system ethanol-water (95:5, v:v) at 293' K and 77' K. T is the absolute temperature,kexcFt. is the exciting wavelength,hf is the maximumof fluorescence emission,Qf is the quantum yield of fluorescence, h is the maximumof phosphorescence emission, Q is the quantum yield oP phosphorescence, and Tpis the phosphorescence lffetime. Cont. of Pyridoxal
T
x excit.
(w)
Qf
h-d
5 x 1o-5 _M 5 x 10-5
293
290
330
0.03
77
290
315
0.40
10-3
77
290
315
x P(W)
420
Q
'Z
0.43
1.2
420
1.2
--Results and Discussion Fluorescence studies of pyridoxal water (95:5, v:v)
in the mixed solvent system ethanol-
were conducted at 293' K and 77O K.
cence characteristics
of pyridoxal
Table I shows the fluores-
at the temperatures examined. At 293' K,
the emission spectrum showsa maximumat 330 w which corresponds to the absorption maximumat 290 w. is shifted increased.
At 70'
K,
the band position of the emission spectrum
towards shorter wavelengths and the fluorescence yield When the phosphorescence spectrum of pyridoxal
is ten-fold
was examined (Fig.
it was found that the long lived emission band is devoid of vibrational and exhibits
structure
a maximumat 420 mu. The observed phosphorescence decay is an
630
l),
Vol. 32, No. 4, 1968
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
X (mp)
Wave
number
(cn?)
x lo4
Fig. 1. Phosphorescence spectra of pyridoxal, , and benzophenone, - - - - -, in ethanol-water glass (95:5, v:v) at 77O K. Both samples had an absorbancp of 0.10 in 1 cm. cuvettes at the exciting wavelength (290 w).
exponential
process that can be described by the expression: I
= IO eFkt
where I is the phosphorescence intensity
at time t and k is the rate constant
for phosphorescence deactivation. The kinetics
of phosphorescence decay is strictly
p = 1.2 seconds) is concentration -3 5 x low5 M to 10 M, the highest concentration lifetime
(z
together with the finding
tested.
This observation taken
that the phosphorescence,spectrum is structureless
Hence, one can disregard competitive
known to be responsible for deviations
(1)
in the long lived emission
bimolecular processes which are
from exponential
of these luminescence studies, the following proposed for pyridoxal:
and the
independent over the range
suggests that only one metastable state is involved process.
exponential
kinetics.
arrangement of energy levels is
a lowest singlet excited
state (fluorescence)
corresponding to an energy of 90 Kcal/mole and (2) a triplet
631
On the basis
excited state
Vol. 32, No. 4, 1968
characterized this triplet
ing to
Am
at 77' K. function
AND BIOPHYSICAL RESEARCH COMMUNIC4‘
by an energy of 69 Kcal/mole. state (fl+
The triplet resonance.
BIOCHEMICAL
The phosphorescence originates
Thus the triplet
The variation
was also characterized
state of pyridoxal
= + 2 transitions
by electron
spin
yields an esr signal correspond-
at 1427 gauss in ethanol water (95:5, v:v)
of the amplitude of the esr signal for pyridoxal
of the excitation
(Fig. 2),.
resembles the absorp-
The energy level of the triplet
(69 Kcal/mole) as well as the long phosphorescence lifetime suggest that the electronic
excitation
energy of pyridoxal
(z
state p = 1.2 seconds)
can be transferred
A (mp) 250
8
,
300 ,
I
I
1
,
,
,
,
1 1
400 ,
g 0 t
\ \ 4.0
as a
wavelength is given in Fig. 2, where it may be seen
that the wavelength dependence of the ear signal strongly K
in
We transition).
state of pyridoxal
tion spectrum at 77*
IONS
3.5 Wave
3.0 number
kn?)
2.5
x lo4
Fig. 2. (Top) Triplet esr si al intensity at 1427 gauss for pyridoxal (10: r M) as a function of excitation wavelength at 77O K. (Bottom) Absorption spectrum, , and fluorescence spectrum (exciting at 290 q~), - - - - -, of 10~~~ M pyridoxal in ethanol-water (95:5, v:v) glass at 77' K.
632
Vol. 32, No. 4, 1968
BIOCHEMICAL
to a suitable acceptor.
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
To test this hypothesis,
the mixture pyridoxal-l-
kynurenine was chosen for the luminescence studies.
L-kynurenine displays an
absorption yield of the sameorder of magnitude as pyridoxal has very low absorbency at 290 nyr.
Pyridoxal,
at 77' K, but it
on the other hand, shows a
maximumof absorption at 290 mc~and a phosphorescence maximumat 420 q. When -4 -4 the mixture pyridoxal (2 x 10 M), L-kynurenine (5 x 10 M) was excited at 290 q,
and the luminescence recorded over the spectral
was found that while the phosphorescence of pyridoxal
range 300-550 w,
it
is decreased, a strong
emission band was detected in the spectral region coinciding with the fluorescence of L-kynurenine.
In addition,
was decreased as the concentration
the phosphorescence lifetime
of pyridoxal
of L-kynurenine was increased (Table II).
Table II Energy transfer from pyridoxal to L-kynurenine The phosphorescence lifetime of pyridoxal (y',) was measured at 390 w with excitations at 290 lq.l.
Pyridoxal Concentration
L-Kynurenine Concentration
2 x 1O-4 M
0
7,
(seconds) 1.2
2x10 -4 M
3 x 1o-4 M
1.0
2x10 -4
5x10 -4 M
0.8
6x10 -4 M
0.7
M
2x10 -4 M
These experiments seemto indicate (triplet-singlet)
is operative,
that a radiationless
since the extensive
cence spectrum of the donor (pyridoxal) acceptor (L-kynurenine) less energy transfer
transfer
overlap between the phosphores
and the absorption spectrum of the
meets the requirements of Forster's
(Porster,
1959).
mechanism
The possibility
633
theory of radiation-
that the efficiency
Vol. 32, No. 4, 1968
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
of energy transfer
is enhanced by complex formation between pyridoxal
L-kynurenine is being investigated
and
in our laboratory.
Acknowledgments. The authors are indebted to Dr. J. J. Ten Bosch of the Oak Ridge National Laboratory for the esr measurements.
References Anderson, F. J. and Martell,
A.
Churchich, J. E. (1967) J. Biol. Forster, Giltmore, 23, 399. Metzler,
T.,
E. (1964) J. Amer. Chem. Soc.2, Chem. 2&
44%.
(1999) Discussions Faraday Sot. 27, 1.
E. H., Gibson, G. E. and McClure, D. S. (1955) J. Chem. Phys. D. E. and Snell,
E. E. (1955) J. Amer. Chem. Sot. 77, 2431.
Ten Bosch, J. J., Rahn, R. O., Longworth, J. W. and Shulman, R. G. (1968) Proc. Natl. Acad. Sci. U.S. 59, 1003. Williams, 2, 56.
715.
V. R. and Neilands, J. B. (1954) Arch. Biochem. Biophys.
634
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
THE TRIPLET
STATE OF PYKIDOKAL*
John E. Wampler+
and Jorge
E. Churchich
Department of Biochemistry University of Tennessee Knoxville, Tennessee
Received
July 8, 1968
The absorption structures
of pyridoxal
laboratories
(Williams
and Martell,
1964).
known about
the
is
also
In spite
luminescence
detected
state
1954;
is
Metzler
and Snell,
of pyridoxal It
of pyridoxal
spectroscopy.
is
from the
triplet
1955;
at
Anderson little
low temperature
the purpose
is
in several
studies,
originates Evidence
of the
investigated
spectrophotometric
properties
transferred
distribution
have been
and phosphorescence.
by esr
energy
solution,
of these
the phosphorescence
excitation singlet
in aqueous
as the relative
and Neilands,
such as fluorescence show that
as well
spectra,
presented state
(77”
of this
in a triplet
is
note
to
state
that
K),
which
electronic
of pyridoxal
to the
of L-kynurenine.
Methods. Luminescence assembly
adopted
A rotating
shutter
phosphorescence recorded
for
(Aminco)
on a Tektronix
as a standard
work
for
in our
of the
samples.
532 oscilloscope
quantum
fellow
yield
conducted
The decay
was 0.1 second.
calculations
1966-69.
629
(Giltmore
Energy
Housing
(Churchich,
was desired
and the most
by the Atomic
in a Dewar
spectrofluorometer
was used when it
decay measurements
was supported
+ NDEA predoctoral
at 77’ K were
observations
properties
of phosphorescence
* This
measurements
to examine
1967). the
of phosphorescence probable
error
Benzophenone et al.,
Conmission,
grant
was in a group
was used
1955).
AT(40-l)-3553.
Vol. 32, No. 4, 1968
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
The esr measurementswere made with a Varian V 4502-12 spectrometer operating at 9.235 GHs. Illumination (Philips)
was provided by 500 watt high pressure mercury arc
and the wavelengths selected by a 500 mmBausch and Lombmonochromator.
Further details
on this apparatus are given by Ten Bosch z
al.,
1968. Low
temperature absorption spectra were obtained on a Perkin-Elmer Model 450 spectrophotometer.
Table I Luminescence characteristics of pyridoxal in the mixed solvent system ethanol-water (95:5, v:v) at 293' K and 77' K. T is the absolute temperature,kexcFt. is the exciting wavelength,hf is the maximumof fluorescence emission,Qf is the quantum yield of fluorescence, h is the maximumof phosphorescence emission, Q is the quantum yield oP phosphorescence, and Tpis the phosphorescence lffetime. Cont. of Pyridoxal
T
x excit.
(w)
Qf
h-d
5 x 1o-5 _M 5 x 10-5
293
290
330
0.03
77
290
315
0.40
10-3
77
290
315
x P(W)
420
Q
'Z
0.43
1.2
420
1.2
--Results and Discussion Fluorescence studies of pyridoxal water (95:5, v:v)
in the mixed solvent system ethanol-
were conducted at 293' K and 77O K.
cence characteristics
of pyridoxal
Table I shows the fluores-
at the temperatures examined. At 293' K,
the emission spectrum showsa maximumat 330 w which corresponds to the absorption maximumat 290 w. is shifted increased.
At 70'
K,
the band position of the emission spectrum
towards shorter wavelengths and the fluorescence yield When the phosphorescence spectrum of pyridoxal
is ten-fold
was examined (Fig.
it was found that the long lived emission band is devoid of vibrational and exhibits
structure
a maximumat 420 mu. The observed phosphorescence decay is an
630
l),
Vol. 32, No. 4, 1968
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
X (mp)
Wave
number
(cn?)
x lo4
Fig. 1. Phosphorescence spectra of pyridoxal, , and benzophenone, - - - - -, in ethanol-water glass (95:5, v:v) at 77O K. Both samples had an absorbancp of 0.10 in 1 cm. cuvettes at the exciting wavelength (290 w).
exponential
process that can be described by the expression: I
= IO eFkt
where I is the phosphorescence intensity
at time t and k is the rate constant
for phosphorescence deactivation. The kinetics
of phosphorescence decay is strictly
p = 1.2 seconds) is concentration -3 5 x low5 M to 10 M, the highest concentration lifetime
(z
together with the finding
tested.
This observation taken
that the phosphorescence,spectrum is structureless
Hence, one can disregard competitive
known to be responsible for deviations
(1)
in the long lived emission
bimolecular processes which are
from exponential
of these luminescence studies, the following proposed for pyridoxal:
and the
independent over the range
suggests that only one metastable state is involved process.
exponential
kinetics.
arrangement of energy levels is
a lowest singlet excited
state (fluorescence)
corresponding to an energy of 90 Kcal/mole and (2) a triplet
631
On the basis
excited state
Vol. 32, No. 4, 1968
characterized this triplet
ing to
Am
at 77' K. function
AND BIOPHYSICAL RESEARCH COMMUNIC4‘
by an energy of 69 Kcal/mole. state (fl+
The triplet resonance.
BIOCHEMICAL
The phosphorescence originates
Thus the triplet
The variation
was also characterized
state of pyridoxal
= + 2 transitions
by electron
spin
yields an esr signal correspond-
at 1427 gauss in ethanol water (95:5, v:v)
of the amplitude of the esr signal for pyridoxal
of the excitation
(Fig. 2),.
resembles the absorp-
The energy level of the triplet
(69 Kcal/mole) as well as the long phosphorescence lifetime suggest that the electronic
excitation
energy of pyridoxal
(z
state p = 1.2 seconds)
can be transferred
A (mp) 250
8
,
300 ,
I
I
1
,
,
,
,
1 1
400 ,
g 0 t
\ \ 4.0
as a
wavelength is given in Fig. 2, where it may be seen
that the wavelength dependence of the ear signal strongly K
in
We transition).
state of pyridoxal
tion spectrum at 77*
IONS
3.5 Wave
3.0 number
kn?)
2.5
x lo4
Fig. 2. (Top) Triplet esr si al intensity at 1427 gauss for pyridoxal (10: r M) as a function of excitation wavelength at 77O K. (Bottom) Absorption spectrum, , and fluorescence spectrum (exciting at 290 q~), - - - - -, of 10~~~ M pyridoxal in ethanol-water (95:5, v:v) glass at 77' K.
632
Vol. 32, No. 4, 1968
BIOCHEMICAL
to a suitable acceptor.
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
To test this hypothesis,
the mixture pyridoxal-l-
kynurenine was chosen for the luminescence studies.
L-kynurenine displays an
absorption yield of the sameorder of magnitude as pyridoxal has very low absorbency at 290 nyr.
Pyridoxal,
at 77' K, but it
on the other hand, shows a
maximumof absorption at 290 mc~and a phosphorescence maximumat 420 q. When -4 -4 the mixture pyridoxal (2 x 10 M), L-kynurenine (5 x 10 M) was excited at 290 q,
and the luminescence recorded over the spectral
was found that while the phosphorescence of pyridoxal
range 300-550 w,
it
is decreased, a strong
emission band was detected in the spectral region coinciding with the fluorescence of L-kynurenine.
In addition,
was decreased as the concentration
the phosphorescence lifetime
of pyridoxal
of L-kynurenine was increased (Table II).
Table II Energy transfer from pyridoxal to L-kynurenine The phosphorescence lifetime of pyridoxal (y',) was measured at 390 w with excitations at 290 lq.l.
Pyridoxal Concentration
L-Kynurenine Concentration
2 x 1O-4 M
0
7,
(seconds) 1.2
2x10 -4 M
3 x 1o-4 M
1.0
2x10 -4
5x10 -4 M
0.8
6x10 -4 M
0.7
M
2x10 -4 M
These experiments seemto indicate (triplet-singlet)
is operative,
that a radiationless
since the extensive
cence spectrum of the donor (pyridoxal) acceptor (L-kynurenine) less energy transfer
transfer
overlap between the phosphores
and the absorption spectrum of the
meets the requirements of Forster's
(Porster,
1959).
mechanism
The possibility
633
theory of radiation-
that the efficiency
Vol. 32, No. 4, 1968
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
of energy transfer
is enhanced by complex formation between pyridoxal
L-kynurenine is being investigated
and
in our laboratory.
Acknowledgments. The authors are indebted to Dr. J. J. Ten Bosch of the Oak Ridge National Laboratory for the esr measurements.
References Anderson, F. J. and Martell,
A.
Churchich, J. E. (1967) J. Biol. Forster, Giltmore, 23, 399. Metzler,
T.,
E. (1964) J. Amer. Chem. Soc.2, Chem. 2&
44%.
(1999) Discussions Faraday Sot. 27, 1.
E. H., Gibson, G. E. and McClure, D. S. (1955) J. Chem. Phys. D. E. and Snell,
E. E. (1955) J. Amer. Chem. Sot. 77, 2431.
Ten Bosch, J. J., Rahn, R. O., Longworth, J. W. and Shulman, R. G. (1968) Proc. Natl. Acad. Sci. U.S. 59, 1003. Williams, 2, 56.
715.
V. R. and Neilands, J. B. (1954) Arch. Biochem. Biophys.
634