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M e m b r. C el l B i o l . , 19 9 8 , × î 1. 1 2 ( 5 ) , ð ð . 6 6 5 — 6 9 0
R e p r i n t s a v a i l a b l e d i r e c t l y f r o m t h e p u b l i sh e r
P h o t o c o p y i n g p e r m i t t e d b y l i c e n se o n l y Î 1 9 9 8 O P A ( O v e r s e a s P u b l i s h e r s A s so c i a t i o n )
N .v . P u b li s h e d b y l i c e n se u n d e r t h e H a r tv o o d
A c a d e m i c P u b l i s h e r s i m p r i n t , p a r t o f
T h e G o r d o n a n d B r e a c h P u b l i s h i n g G r o u p
P r i n t e d i n I n d i a
Singlet M olecular Oxygen in Photobiochemical
System s: I R Phosphor escence Studies
À . À . Krasnovsky, .1Ã.
Department î~Â1î 1îäó, Moscow State University, Vorobievy á î ãó, Moscow 117899
Bakh Institute î /'Biochemistry, Russian Academy î/'Þñ!åï ñåõ, 33 Leninskii Prospect,
Moscow i i 707I
/àõ: (095) 954-2732
Singlet molecular oxygen ( 0 2) is one of the most active intermediates involved in
photosensitized oxygenation reactions in chemical and biological systems. Deacti-
vation of singlet oxygen is accompanied by infrared phosphorescence (1270 nm)
which is widely employed for 0 2 detection and study. This review considers
techniques for phosphorescence detection, phosphorescence spectra, quantum yields
and kinetics under laser excitation, the radiative and real 0 2 lifetimes in organic sol-
1
vents and water, 0 2 quenching by biomolecules, and estimation of singlet oxygen 1
lifetimes, diff usion lengths and phosphorescence quantum yields in blood plasma,
cell cytoplasm, erythrocyte ghosts, retinal rod outer segments and chloroplast thyla-
koids. The experiments devoted to 0 2 phosphorescence detection in photo-
sensitizer-containing living cells are discussed in detail. Information reviewed is
important for understanding the mechanisms of photodestruction in biological
systems and various applied problems of photobiology and photomedicine.
(Received á M ay, 1997)
In this nårnoï àl issue dedicated to my father it is worth noting that though
photosensitized oxygenation of organic compounds (photodynamic
reactions) has been known since the beginning of the 20th century, its mole-
cular mechanisms had been à subj ect of heated discussions for à very long
time [1, 2]. Ì ó father began his w ork in science w ith investigation of such
reactions. In the first papers photosensitizers were titanium dioxide and later
phthalocyanines and chlorophyll. Substrates w ere oleic and ascorbic acids [3,
4]. A s à result, reversible chlorophyll photoreduction by ascorbic acid
(K rasnovsky reaction) and chlorophyll-photosensitized electron transfer
were experimenta
lly observed [5] . Those studies became à basis for
understanding the primary mechanisms of photosynthesis. Besides, they
665
666
À. À. KRASNOVSKY, Jr.
showed that triplet-state mediated porphyrin photoreduction can be à primary
stage of photodynamic reactions of porphyrins and phthalocyanines.
According to the modern classification, primary events of photodynamic
reactions correspond to "type 1" or "type 2" mechanisms [6]. Photodynamic
reactions based on primary porphyrin photoreduction by the oxidizing
compound are an example of à type 1 mechanism where molecular oxygen
plays the role of an electron acceptor:
Ð+ ~ = 'Ð~ — 'Ð* + 1 = Ð- + ð +
'Ð + O~ Ð+ 'Î ~ ,
where Ð, 'P~ and ÇÐ' are photosensitizer molecules in the ground, excited
singlet and triplet states, and D is à substrate of photooxygenation. In the
alternative "type 2" mechanism, the primary event is the energy transfer from
triplet photosensitizers to dioxygen with population of its singlet 'Ü state
('O~); then, singlet oxygen oxidizes appropriate substrates [6]:
Ð + hv — ~ 'Ð — + Ð + 0 ~ '0 ~ + Ð
~ 2 D = Dî õ
Both reaction types are widespread. Along with oxygenation of organic
compounds in chemical systems, these reactions determine photoinhibition
î ÃðÜî 1î üóï é åÿ â and photodestruction î ÃÍ å photosynthetic apparatus under
strong light [6 — 8], participate in the photodamage of eye retina and
crystalline lens [9 — 11], infl uence phototaxis of microorganisms [12], account
for phototoxicity of various drugs [13], are the cause of the photodestructive
action of porphyrins at their excess accumulation in plant and animal cells
[14, 15], determine photodynamic therapy of malignant tumors [15 — 17], are
successively used in DNA cutting and virus destruction [18, 19]. Singlet
oxygen plays à special role. On the one hand, it participates in numerous
photosensitized reactions, on the îáæåã hand, the extent of its participation is
an indicator î é Üåê mechanism.
In 1976 this author found that photosensitized 'O~ generation in
air-saturated solutions was accompanied by phosphorescence at 1270 nm
corresponding to radiative deactivation of the dioxygen 'Ë state. Simulta-
neously short-wavelength delayed fl uorescence was detected in the visible
spectral region, whose intensity was proportional to the squared 'Oz
concentration [20 — 23]. As shown recently, short-wavelength fl uorescence is
emitted by dimers (dimoles) (~Ü )~, by the iEg state of molecular oxygen
(760 nm) [24 — 26], and by the dyes capable of accepting the energy of two
'0 ~ molecules [24, 27, 28]. Delayed fl
uorescence of certain dyes is so
PHOSPHORESCENCE STUDIES OF SINGLET OXYGEN 667
intensive that might be à promising method for 'Oz detection and investi-
gation [28, 29]. However, so far the measurement of 1270 nm phospho-
rescence is considered the most reliable method for 'Oz registering and
analysis [30 — 32]. The present paper is à short survey of measurement
techniques, mechanisms and parameters of 'O> IR-phosphorescence, and of
the results of its use in photochemical and photobiochemical experiments.
The survey is based on studies of the laboratory of this author and on à
comparison of parallel studies by îáæåã groups.
1 M ethods of Phosphorescence M easurements
IR phosphorescence of molecular oxygen is inconvenient for optical
measurements because its quantum yield is low and standard photometric
equipment (photomultipliers and semiconductor detectors) has à low signal-
to-noise ratio at 1270 nm. For this reason, à series of original devices were
created that can be attributed to two maj or groups: those with steady-state
(modulated) excitation and signal detection and those designed for time-
resolved measurements.
Our equipment was described in [33]. As photodetectors, we used FEU-83
photomultipliers of S-1 spectral response cooled to — 60' . The first measure-
ments of photosensitized 'Oz phosphorescence in air-saturated solutions
were performed using our set-ups with mechanical phosphoroscopes which
allowed the measurement of emission and excitation spectra, quantum yields
and lifetimes of phosphorescence whose lifetime exceeded 500 iis. The
next-generation set-ups had ï î phosphoroscopes and measurements were
made using the mode î é ëå steady-state spectrofl uorimeter with excitation by
stationary light sources. More recently we have constructed à time-resolved
photon counter with accumulation and averaging of the phosphorescence
signal é.î ò numerous laser fl ashes. This allows the signal-to-noise ratio to be
increased and the phosphorescence kinetics and time-resolved phospho-
rescence spectra to be measured. In the first set-ups of this type, time
resolution was limited by multichannel analyzer dwell time which was equal
to 5 lis in our NTA-1024 analyzer. Due to cooperation with the Laser Center
î Ãthe Moscow State University we considerably improved this technique by
using à copper vapor laser with à pulse frequency of about 10 kHz and time-
correlated single photon counting technique (Fig. 1). Using this apparatus wå
achieved the highest time resolution available for our photomultiplier, about
15 ns, without à decrease in sensitivity. At present, we use à much more
reliable copper vapor laser "Femta" of newer generation, manufactured by
the Lebedev Physical Institute of the Russian Academy of Sciences.
In our j oint projects with US laboratories, measurements were carried out
with germanium photodetectors of Northcoast Corporation (Santa Rosa,
USA), cooled by liquid nitrogen. An interference filter of the appropriate
66 8 À . À . K R A SN O V SK Y , Jr
I C opper v apor laser ',
D iff use l ight
PM T -"start"
pream plif ier
)D iscr im inator (
1 Òèï å-am plitude I .
converter
1 L ight filter L , à .è
( M onochrom ator )
- stop ( - )
pream lif ier
p iscrim inator )
N 7A - 1024
~ m u lt ic h an n el
an aly z er
~ Pr in t er ~- C o m p ut er 1 1 1 122 0 1320 14 20 1520 162 0 nm
F ig . 1 F ig . 2
F igu r e 1. B l ock -schem e of th e set -up f or m easur em ent o f photo sensitiz ed
si ng let ox y gen ph osph or escence w ith nano seco nd resolut ion usin g p h ot o-
m u ltip li ers (PM T ) an d techn iqu es o f t im e-co rrelat ed sing le-ph oton c ou ntin g
[33 , 4 3, 4 4] .
F ig u r e 2 . Sp ect r um of p hoto sen sit ized sing let ox yg en ph osp horescen ce in
air-satu rated so lu tion of t etr aph eny l porphy r in in Ñ Ñ 14. I . is the
ph o sph or esc en ce inten sity [3 2, 3 8, 39 ] .
w av e len g t h w as p la ce d in fr o n t o f th e p h o t o d i o d e . E x c ita ti o n w a s p r o d u c ed
b y an N d — Y a g l aser , m a i n l y b y t h e 5 3 2 n m b an d [2 7 — 2 9 , 1 19 an d r ef s
th er ein ] . T h e sig n a l î Ã th e p h o to d i o d e w a s r eg i st er ed b y à d ig it al o sc il l o sc o p e
w h i ch p er m itt ed ac c u in u l at i o n an d av er ag in g o f t h e si g n a l f r o m n u m er o u s
la ser fl ash e s . T h e se set - u p s ar e c on v en i en t i n u se . T h e ir t im e r eso lu t io n w as
l im it ed b y t h e p h o t o d io d e r esp o n se ( sev er al m ic r o se c o n d s) .
2 M ec h a n i sm o f D i o x y g en E x c it a t i o n a n d P h o sp h o r esc en c e S p ec t r a
(F i g . 2 )
T h e m o st c om m o n m ec h an i sm o f p h o t o sen sit i ze d ' 0 > p h o sp h o r esc en c e i s as
f o l lo w s:
+~ 2
P + J ~ 1P e ~, Çð å ~~ 10 + P
' 0 2 — ~ 0 2 + Itv ( 12 7 0 ø ï ) .
T h e g a s p h a se p h o sp h o re sc en c e sp ec tr u m h as à m aj o r m ax im u m at 12 6 8 n m
( ' Ü ( Î ) ~ ~Õ (Î ) tr an sit io n ) an d 3 r o tat i on a l sh o u l d er s P , Q an d R [ 3 4 ] . O u r
4
PHO SPH ORESCEN CE STUD IES OF SIN GL ET OX Y GEN 669
experiments showed that in ÑÑ1Û this maximum is shifted towards 1274+2
nm, the band halfwidth being 18+1 nm [35]. The subsequent more precise
measurements, in particular, the recent data obtained using Fourier
spectrometers (resolution 4 cm ') indicated that the maj or maximum varied
from 1270 to 1279 nm in diff erent solvents. The band halfwidth was 15 — 19
nm (87 — 120 cm '), the rotational structure was not revealed [36, 37]. Along
with the maj or band, à 50 times wåakåã maximum was observed at 1590 nm
which corresponded to à 'À (Î ) ~ ~~ (1) transition [38, 39]. Besides, à series
of even less intensive bands corresponding to the transitions involving vibra-
tional sublevels of solvent ò î 1åñè1åû ÷åãå found at 1350 — 1450 nm [40] and
1070- 1170 ï ò [25].
3 Phosphorescence K inetics in O rganic M edia
Phosphorescence kinetics after à short laser fl ash is described by the equation
similar to the well-known formula for the time course of two consequent
monomolecular reactions:
L(t) = ~' " [ åõð ( — ~l ò, ) — åõð ( — tlat,)] , (3 .1)
where /ñ,,„ is the rate constant of energy transfer from Ð to Oz resulting in
'Oz generation, ò, is the 'Oz radiative lifetime; òä is the real 'Oz lifetime, ~, is
the Ð* lifetime; [ Ð ]î is the concentration of triplet photosensitizer mole-
cules j ust after laser fl ash; [Oz] is the dioxygen concentration in solution [32,
41 — 44, 61]. Analysis î é Û equation shows that the kinetic curves have two
phases: rise and decay. The rise is conditioned by the rate of 'Oz formation
caused by energy transfer from ~P~ to 0 ~, the decay is determined by 'Oz
deactivation. When the rise is changed by the decay à peak is observed on the
kinetic curves. The time interval between this peak and à laser fl ash (~,„,„) can
be described as:
lï (ò„/x,)
tmax
max (3 .2 )
In air-saturated organic solvents ò, = 250 — 350 ns, which is less than òä by
several orders of magnitude. In this case, the peak is achieved 1 — 3 ðü after
the laser fl ash, and 2 — 3 lis later the decay becomes monoexponential with the
lifetime corresponding to òä. The first phosphorescence òä measurements
were carried out in our laboratory on à set-up supplied with à
6 70
À . À . K R A SN O V SK Y , 1ã.
1.6
0.8
0.6
1.2 0.4
0.2
0
Òèï å, it s
0 .4
1 1 1 1 I I I 1
20
Òèï å, p s
40
Figure 3. K inetics of photosensitized singlet oxygen phosphorescence under
excitation by pulses of the copper vapor laser (13 kH z) in air-saturated
solution of pheophytin à in benzene. Z ero on the time scale corresponds to
the end of laser fl ash. The trace 1 (decay kinetics) w as obtained as à result of
signal averaging over áõ 105 laser fl ashes, 1 channel corresponded to 140 ns.
The trace 2 (rise kinetics) w as obtained after 1.2õ 10~ laser fl ashes, 1 channel
corresponded to 7 ns. Solid line show s the data approximation by the
equation 3.1. [E.À . Bashtanov and À .À . K rasnovsky Jr., 1998].
phosphoroscope. W e found that in Ñ Ñ 14, ò~ w as about 30 m s [2 1 — 23] w h ich
is 40 tim es greater than the v alue obtain ed earlier in th e ex per im ents w ith
ch em ical traps of sing let oxy gen . Subsequent studies by î áæåã lab oratories
con f irm ed th is v alu e [4 5 — 47] . A s show n later, in h igh ly pur if ied Ñ Ñ 14 òä is
even h igher, 87 m s. H igh ò~ v alu es (2 5 — 100 m s) w ere also observ ed in î áæåã
solvents w h o se m olecules did not hav e hy dr ogen atom s [46] . It w as
establ ished using set-ï ð û ã laser ex citation that t a = 10 — 300 )i s in organic
solv ents contain ing hy drogen atom s. R ep lacem ent of hy drog en by deuterium
led to à 20 — 304 î 16 increase of ~~ [38, 4 5, 46, 4 8] .
T he phosphorescence rise phase can be read ily observed at low ered oxy gen
pressure w hich caused à decrease of the decay rate of the photosensitizer
trip let states and sh ifted th e peak tow ards longer tim es [42] . M odern equ ip-
m ent also allow s reliable ri se reg istrat ion in air- saturated so lv ents. T he p eak
position fits eqs. (3 .1) and (3.2) (Fig. 3).
M erk el and K earn s suggested that ò~ v alues ar e determ ined by the
efficien cy of ' 0 2 quench ing w h ich results from the energy transfer fr om ' 0 2
to v ibr ation al sublev els of solv ent m olecules [49] . In order to estim ate the rate
PHOSPHORESCENCE STUDIES OF SINGLET OXYGEN 671
constants of this process, we studied the quenching of photosensitized 'Oz
phosphorescence in ÑÑ1Û by microamounts of the solvents whose molecules
contained hydrogen atoms [35, 50, 51]. We found that the quenching obeyed
the Stern — Volmer equation:
(<z)o/9 a)q = (~ä)î /(~ä)ä = 1 + >q(~, )o~q (3 .3)
where (L~)p, (7a)p, (Ñä), and (òä), are the 'O> phosphorescence intensities
and 0 2 lifetimes in ÑÑ1Û in the presence and absence of the quencher. The
À, values for solvent molecules were close to those previously obtained in the
gas phase. Then, we noticed that if one rewrites the Stern — Volmer equation
in the following form:
1/(~a)q = 1/(t )p + À Ñ~
and set that Cq Ñ,„,„, where » is the molar concentration of à neat
solvent used as à quencher, we obtain that À Ñ,1» 1/(òä)~ and hence:
~ä = 1/À,„Ñ (3 .4)
where t is the singlet oxygen lifetime in the neat solvent [35, 50, 51]. Experi-
mental analysis showed that eq. (3.4) is really valid (Table 1). À similar
conclusion was made in parallel studies by Hurst and Schuster [52] and
Rodgers [53]. Thus, these data indicate that the lifetime of singlet oxygen is
caused by 'Oz quenching by solvent molecules, therefore, when one knows
À values, t can be determined by eq. (3.4) without recourse to time-resolved
kinetic measurements.
4 Phosphorescence K inetics in Aqueous Solutions
First measurements of photosensitized 'O~ phosphorescence in D>O,
mixtures of DzO and HzO and in neat HzO were performed on the set-ups
with stationary excitation [54, 55]. The first kinetic measurements under laser
excitation were described in [41, 48, 56 — 58]. The exponential decays were
observed with the lifetimes 56 — 68 ðü 1ï DzO and 3.1 — 3.9 ps in HzO. Modern
equipment allows reliable measurements of the rise, peak and decay of the
kinetic curves. Since oxygen solubility in water is 10 times less than in
organic solvents, the rise proceeds slower in aqueous media. In D>O with
tetra-(p-sulfophenyl) porphyrin (TPPS) as à photosensitizer, à peak was
observed 7 — 8 Its after à laser fl ash. According to eqs. (3.1) and (3.2), this
peak position corresponds to x, = 2.7 Iis [32, 59, 60] (Fig. 4). It is noteworthy
6 7 2
À . À . K R A SN O V SK Y , 1ã.
T a b le 1. C om p ari son î é ë å ex p er im ental v alues of òä an d 1/(I~, Ñ , „ ) [3 1, 3 2, 3 5, 50] .
S o l v e n t s kq, Ì s 1/(AC~~~~ ), p,s Òä , ð,~
C h l o r o f o r m
è - H e p t a n e
n - D o d e c a n e
D i e t h y l e t h e r
A c e t o n e
B e n z e n e
P y r i d i n e
1 - D e c a n o l
E t h a n o l
M e t h a n o l
T r i t o n Õ - 1 0 0
E g g l e c i t h i n 1 2 . 4 6 . 8 4 . 4 9 . 6 1 3 . 7 1 1 . 2 1 2 . 4 5 . 2 1 7 . 1 2 4 . 7 1 . 6 4 1 . 6 3 8 0 6 7 0 0 1 1 0 0 0 4 3 0 0 1 5 0 0 3 6 0 0 4 4 0 0 1 2 0 0 0 5 4 0 0 4 3 0 0 5 3 0
1 0 ' 2 1 0 2 2 2 1 2 4 4 9 2 5 1 8 1 6 1 1 9 . 5 1 1 7 2 5 0 2 8 2 5 3 5 5 1 3 1 1 6 1 8 1 5 1 0 . 5 1 2
~ Ñ „ , v a l u e s w e r e c a l c u l a t e d b y d i v i d i n g t h e s o l v e n t d e n s i t y ( g r a m / d m ~ ) b y t h e s o l -
v e n t g r a m - ò î 1 . T h e I ~, v a l u e s w e r e m e a s u r e d b y q u e n c h i n g 0 2 p h o s p h o r e s c e n c e i n 1
C C 1Û w i t h m i c r o a m o u n t s o f s o l v e n t s . T h e ÷ ä v a l u e s w e r e d e t e r m i n e d f r o m t h e k i n e t i c s
o f p h o s p h o r e s c e n c e d e c a y o n s e t - u p s w i t h l a s e r e x c i t a t i o n .
t h a t i n D 2 O ~ ä s t r o n g l y d e p e n d s u p o n t h e d u r a t i o n o f s t o r a g e . I n s o l u t i o n
p r e p a r e d w i t h D 2 O t a k e n f r o m t h e c o n t a i n e r o p e n e d j u s t b e f o r e u s e , ò ä w a s
a b o u t 7 0 p s . A f t e r à 1 - h s t o r a g e i n t h e p r e s e n c e o f a i r , ò ä d e c r e a s e d t o 5 0 — 6 0
l i s . 1 . o n g s t o r a g e r e s u l t e d i n t h e d e c r e a s e î Ã ò ä t o 3 5 — 4 0 I i s . I t i s l i k e l y t h a t ò ä
r e d u c t i o n i s c a u s e d b y H 2 O a c c u m u l a t i o n [ 3 2 ] .
T h e f i r s t o b s e r v a t i o n o f t h e r i s e a n d d e c a y o n t h e p h o s p h o r e s c e n c e k i n e t i c
c u r v e i n a i r - s a t u r a t e d H 2 O w a s r e p o r t e d b y P a r k e r [ 4 1 ] . L a t e r , w e p e r f o r m e d
d e t a i l e d k i n e t i c m e a s u r e m e n t s u s i n g à n a n o s e c o n d s p e c t r o m e t e r w i t h
t i m e - c o r r e l a t e d s i n g l e - p h o t o n - c o u n t i n g [ 4 3 , 4 4 , 6 1 ] . T h i s a l l o w e d u s t o
m e a s u r e t h e ' 0 > p h o s p h o r e s c e n c e r i s e s a n d d e c a y s w i t h i n à b r o a d r a n g e o f
o x y g e n p r e s s u r e a n d t e m p e r a t u r e . T h e e x p e r i m e n t s s h o w e d t h a t a t à p r e s s u r e
0 . 2 — 1 5 a t m a n d t e m p e r a t u r e 1 0 — 6 0 ' Ñ t h e d e c a y s c o r r e s p o n d e d t o e x p o -
n e n t i a l s w i t h t h e l i f e t i m e o f 3 . 1 1 0 . 1 p s . 1 ï a i r - s a t u r a t e d T P P S s o l u t i o n s a t
r o o m t e m p e r a t u r e à p e a k w a s o b s e r v e d 2 . 5 ) i s a f t e r à l a s e r fl a s h ( F i g . 5 ) . T h e
r i s e c o r r e s p o n d e d t o t , = 2 . 0 l i s .
5 P h o s p h o r esc en c e Q u a n t u m Y i el d i n t h e S o l u t i o n P h a se
F o r q u a n t i t a t i v e d e s c r i p t i o n î é ë å e ff i c i e n c y î Ã ð Ü î 1 î ç å ï ç é ã å ä ' 0 > p h o s p h o -
r e s c e n c e o n e c a n u s e i t s q u a n t u m y i e l d o r , i n t h e c a s e î Ã ð è 1 ü å l a s e r e x c i t a t i o n ,
t h e i n i t i a l ( z e r o - t i m e ) p h o s p h o r e s c e n c e i n t e n s i t y . T h e q u a n t u m y i e l d ( ~ð 1, )
c o r r e s p o n d s t o a n e q u a t i o n :
PH OSPH ORESCEN CE STU DIES OF SIN GLET OX Y GEN 673
l Î
ô
ÿ 1
ñ
ILI
| ×
50
Òèï å, li s
Figure 4. Kinetics of photosensitized singlet oxygen phosphorescence in
air-saturated DzO, measured by this author on the set-up of the Arizona State
University under excitation by Nd — Yag laser, 532 nm. The signal was
registered by à photodiode of Northcoast Corporation via an interference
filter with the transmission maximum 1270 nm. Photosensitizer was
tetra-(ð-sulfophenyl) porphyrin [119].
mph vawr
( 5 . 1)
where <ðä is the quantum yield of '0 2 generation Úó à photosensitizer, rp, is the
'0 2 phosphorescence quantum yield, ~, is the 'Oz radiative lifetime. The
zero-time intensity (10, in quanta per second) is calculated by extrapolation of
the exponential phosphorescence decay to zero time:
~î = I i~ qpa~ú
( 5 .2 )
where 11„ is the number of quanta absorbed by à photosensitizer during à laser
fl ash. In the gas phase at an oxygen pressure less than 1 atm, the '0 2 decay
corresponds to à highly forbidden intramolecular radiative magnetic dipole
transition w ith ï î contribution of nonradiative processes. Therefore, z, = ~ä =
3845 s and ip, = 100% [34, 62]. W ith the increase of the oxygen pressure or
in the presence of à foreign gas, argon, for example, the radiative lifetime
decreased to several seconds, which results from the transformation of the
'Î 2 electronic structure in '0 2 collision complexes w ith foreign particles
[63 — 65].
6 7 4
À . À . K RA SNOV SK Y, )ã.
1500 » 10
103
10'
'ú | 10
È
< 500
À
' Ú ~ 10 I n L P 1 20 ü Ú lnL +."ë 1250 1300nm ã
10 20 ps 0 5 10 p s
Figure 5. Decay kinetics and spectrum of photosensitized singlet oxygen
phosphorescence in aqueous (Í 20 ) systems: à, air-saturated solution of
tetra-(ð-sulfophenyl) porphyrin, ðÍ 7 after excitation by 10 fl ashes of
copper vapor laser; Ü, air-saturated suspension of yeast cells loaded with
tetra-(ð-sulfophenyl) porphyrin after excitation by Çõ10 fl ashes of copper
vapor laser in à fl ow system. The spectrum corresponds to the gap 2 — 7 Its
after à laser fl ash. L is the phosphorescence intensity [33, 43, 44, 61].
The quantum yields of '0 2 phosphorescence in photosensitizer solutions
were much less than 100% but considerably more than those that one w ould
expect if t, were 3845 s [66 — 71]. The ~, values calculated from the experi-
mental y h values using eq. (5.1) or from the intensities of the dioxygen
absorption bands [65, 71] are shown in Table 2. It is seen that in solutions t,
is markedly less than in the gas phase at à low pressure, but coincides w ith
that for '0 2 collision complexes w ith foreign particles. Hence,
phosphorescence is emitted by solvated '0 2 molecules.
Thus, solvents quench '0 2 which causes à strong decrease of òä and the
phosphorescence yield (Table 1). Simultaneously, solvents decrease ò, (Table
2) which leads to an increase of the phosphorescence yield. Quenching is
much more effective than activation. A s à result, the total phosphorescence
yield in photosensitizer solutions is significantly less than 100% . Table 3
illustrates the y h values in diff erent solvents estimated by this author using
experimental t, and ãä values and eq. (5.1). Òî make calculations wå took into
account that the absolute ~, values reported by diff erent groups do not
coincide (Table 2) whereas the reported relative ò, values are much ò î ãå
consistent [72, 73]. Therefore, we suggested that the most likely t , in benzene
is = 1 s and used literature relative t, values to estimate t , in î ë åã solvents.
Table 3 shows that the phosphorescence quantum yields in hydrogen-atom
containing solvents are 0.02% and less. The lowest y h was found in water,
the natural medium of living organisms. Therefore, singlet oxygen phospho-
rescence in aqueous media is the most diff icult subj ect of studies.
PH O SPH O R E SC EN C E ST U D I E S O F SIN G L E T O X Y G EN 67 5
T a b l e 2 . V a l u e s o f r , i n C C 1 4 a n d b e n z e n e .
M e t h o d o f m e a s u r e m e n t s Ñ Ñ 1 Û Ñ 6 Í 6
R e f åãån cå
A b s o r p t i o n
A b s o r p t i o n
P h o s p h o r e s c e n c e
P h o s p h o r e s c e n c e
P h o s p h o r e s c e n c e
P h o s p h o r e s c e n c e 5 . 7 2 4 + 2 1 . 2 5 + 0 . 3 1 . 0 + 0 . 4 2 5 . 2 ~ 1 . 1 2 Í 0 . 2 2 + 0 . 0 7 0 . 7 + 0 . 2
1 . 8 Û . 7 [ 6 5 ] [ 7 1 ] [ 6 6 , 6 7 , 7 2 ] [ 6 9 , 7 1 ] [ 6 8 ]
[ 7 0 ]
~À solv ent w as h ex afl u orob en zene .
T a ble 3 . Q u antu m y iel d s of ' O z p ho sph oresc en ce in d iff erent solv en ts.
S o l v e n t
C h l o r o f o r m
B e n z e n e
A c e t o n e
M e t h a n o l
Ð ~ Î
Í 2 0 1 . 3 3
1
2 . 6
4 . 8
8 . 3
8.3 ò õ 1Ô , s
2 5 0
3 1
5 1
1 0
6 7
3 . 1 1 . 9 õ 1 0 4
3 . 1 õ 1 0 5
2 x 1 0 ~
2 . 1 õ 1 0 6
8 1 õ 1 0 - ü
3 . 8 õ 1 0 ~
~R elativ e v alues î È , ar e t ak en fr om [73] .
E q u a t i o n ( 5 . 2 ) a n d T a b l e 3 a l s o s u g g e s t t h a t t h e i n i t i a l p h o s p h o r e s c e n c e
i n t e n s i t y ( 1 0 ) i s s i g n i f i c a n t l y l e s s d e p e n d e n t o n s o l v e n t s t h a n y h . I n d e e d , < ð 1 ,
d e p e n d s o n x a w h e r e a s 1 0 i s à f u n c t i o n o f ò , w h i c h i s m u c h l e s s s e n s i t i v e t o
s o l v e n t s t h a n ~ ~ . N e v e r t h e l e s s , p r e c i s e m e a s u r e m e n t s î É Ü å p h o s p h o r e s c e n c e
d e c a y s a r e n e e d e d t o o b t a i n a c c u r a t e 1 0 v a l u e s , w h i c h , a s m e n t i o n e d a b o v e ,
a r e c a l c u l a t e d b y e x t r a p o l a t i o n o f t h e e x p o n e n t i a l d e c a y s t o z e r o t i m e .
á S i n g l e t O x y g e n P h o s p h o r e s c e n c e i n B i o l o g i c a l S y s t e m s a n d t h e i r
M o d e l s
S o f a r , e x p e r i m e n t a l o b s e r v a t i o n o f ' 0 > p h o s p h o r e s c e n c e i n l i v i n g t i s s u e s
w a s r e p o r t e d o n l y b y P a r k e r [ 7 4 ] . I n h i s e x p e r i m e n t , a n a r g o n 1 î ï l a s e r w a s
u s e d t o p u m p t h e L e x e l M o d e l 7 0 0 L d y e l a s e r o p e r a t i n g w i t h D C M a s a n
a c t i v e d y e . T h i s p r o v i d e d a n o u t p u t w a v e l e n g t h i n t h e r a n g e î Ã 6 0 0 t o 7 0 0 n m .
F o r m o s t m e a s u r e m e n t s , t h e w a v e l e n g t h w a s f i x e d a t 6 3 0 n m . T h e d y e l a s e r
r a d i a t i o n w a s m o d u l a t e d b y s q u a r e w a v e p u l s e s o f a n a c o u s t o - o p t i c m o d u -
l a t o r . T h e o b j e c t w a s à m o u s e w i t h i m p l a n t e d c a n c e r t u m o r l o a d e d w i t h
P h o t o f r i n I I , a n d à s o l u t i o n î é å 1 ã à ï å é ó 1 p y r i d y l p o r p h y r i n ( T M P P , 1 0 4 Ì )
676
À. À . KRA SNOVSKY, 1ã.
in water phosphate buffer. An average power of laser radiation was 20 mW.
Luminescence was focused by à 1 inch-diameter 25 ò ò focal-length lens
through à system of light filters with the transmission maxima at 1215 or
1283 nm or through à SPEX spectrometer to à germanium detector
(EO-817S, Northcoast Corporation). The photodiode signal was sent to à
lock-in amplifier. The time constant of the registering system was 30 Iis. In
the TMPP solution, photosensitized phosphorescence at 1270 nm was
detected. Dependence of the phosphorescence intensity upon modulation
frequency reached the maximum at 40 kHz which correlated with the ãä and
ò, values (3.2 lis and 2.4 Iis, respectively) obtained for TMPP solution in
separate experiments. In the murine tumor, the phosphorescence spectral
maximum was at 1260 nm and the frequency maximum, at 20 kHz. The latter
indicated that ã, probably increased up to 9.6 lis in the tumor tissue [74].
Patterson et al. tried to repeat Parker's experiment. Photofrin II and the
sulfo derivative of aluminum phthalocyanine were used as photosensitizers.
Dioxygen phosphorescence was observed in methanolic solutions of these
compounds and in an aqueous solution of phthalocyanine. In an aqueous
solution of Photofrin, stained mouse tumor and in water suspension î Ãtumor
cells, phosphorescence was not observed [75].
Firey et al. attempted to find 'Oz phosphorescence in à suspension of
mouse myeloma cells loaded with zinc phthalocyanine. Phthalocyanine was
introduced into cells using dye-containing liposomes. The cell suspension
was prepared in D>O. 1ï the presence of air, phthalocyanine triplets with the
lifetimes of 8 and 52 ðâ were observed. The rate constants of triplet
quenching by oxygen were found to be 5.5õ10â and 8.1õ10~ Ì 's ',
respectively. However, 'O~ phosphorescence was not detected in aerated cell
suspensions though it was readily detected in phthalocyanine-containing
liposome suspensions and detergent micelles in heavy water [76]. À similar
result was reported by Truscott et al. [77]. In their experiments, à fibroblastic
cell line (ÂÍ Ê21) transformed by polyoma virus was loaded with hemato-
porphyrin or its derivative (HPD). After excitation by ruby laser (347 nm) in
the presence of air, porphyrin triplet states with the 7.5 lis lifetime were
revealed. After nitrogen purging the triplet lifetime increased by ò î ãå than
two order
s î Ãmagnitude. This indicates that porphyrin triplets are efficiently
quenched by oxygen in cells. However, the porphyrin loaded cell suspensions
in heavy water showed very weak oxygen phosphorescence which was
ascribed to the photosensitizing action of porphyrins localized extra-
cellularly.
Using our nanosecond laser spectrometer based on time-correlated single
photon counting we succeeded in measuring 'O> phosphorescence in water
(HzO) suspension of porphyrin-loaded yeast cells Saccharomyces vini'. Cells
were suspended in TPPS-saturated 0.1 Ì phosphate buff er (ðÍ 7.2)
containing 0.3 Ì NaC1. After 1-h incubation of cells at 25'Ñ, the suspension
PHOSPHORESCENCE STUDIES OF SINGLET OXYGEN 677
was centrifuged at 800 g. The supernatant was removed, the sediment was
suspended in the buffer solution and centrifuged again. Washing was
repeated 3 — 6 times to obtain à colorless supernatant. The concentration of
porphyrin in the washed sediment was rather high. Then, it was diluted by
buff er to obtain à 250 ml suspension with about 0.15 absorbance at 514 nm
in 1 cm optical path. À fl ask containing 250 ml of the cell suspension was
connected to à 0.7 ml fl ow cuvette installed just in front of the ò î ï î -
chromator entrance slit. Phosphorescence was measured at 1270 nm with à
100 ns delay after à laser fl ash. Àéåã irradiating the suspension with 10 ns
pulses of à copper vapor laser (514 and 578 nm) we observed IR lumi-
nescence which rose for 300 ns after à laser fl ash and then decayed. Two
components were seen on decay traces. The lifetime of the slow component
was about 3 Iis and its spectrum measured in the gap 2 — 7 lis after à fl ash
showed à 1272 nm maximum (Fig. 5). The lifetime î Ãí å fast component was
<1 iis. The spectrum measured in the gap 0.4 — 2 liç after à fl ash had the same
1272 nm maximum. However, at Õ < 1250 øï the luminescence intensity
increased as compared to the slow component. We proposed that the slow
component corresponded to singlet oxygen located in buffer extracellularly,
whereas the fast component was the total of '0 > and porphyrin phospho-
rescence [43, 44]. The origin of the fast decay component and the rapidly
occurring peak on the kinetic curve is still unclear. Kinetic analysis suggests
that this might be if the components existed where the òä and x, values did not
exceed l lis.
Baker and Kanofsky studied Ü1210 leukemia cells stained with the poly-
meric fraction of à hematoporphyrin derivative [60]. À dye laser pumped by
fl ash lamps was used for photoexcitation. The energy of à laser fl ash was 15
mJ, fl ash duration was 300 ns. In cell suspensions in DzO, à luminescence
with two decay phases was observed. The first phase (Î — 2 ðç) corresponded
to polyporphyrin fl uorescence; the second (20 — 90 ms), to 'Oz phospho-
rescence. The second phase decayed biexponentially with the lifetimes of
4.5+0.5 and 49+4 Iis. The slow component had à spectral maximum at 1270
nm and was suppressed by singlet oxygen quenchers: histidine, carnosine and
HzO. The authors suggested that the slow component corresponded to '0 >
which had been generated on the surface î Ãthe tumor ce
lls and then diffused
into the buffer solution. The origin of the fast component remained unclear.
Iï the subsequent studies by the same authors 5-(N-hexadecanoyl) amino-
eosin was used as photosensitizer [78]. This dye is known to accumulate
inside cell membranes. It was shown that photosensitized light emission
detected within the range of 10 — 80 lis after à laser fl ash was 'Oz phospho-
rescence originating from singlet oxygen molecules located in heavy water
outside the cells. However, the phosphorescence intensity in the cell
suspension was less by two orders of magnitude than in solutions of
detergent-solubilized eosin in D>O.
678
À. À. KRASNOVSKY, 3ã.
Phillips er al. studied cells of Porphyromonas gingivalis loaded with
Toluidine Blue. The dye was mostly (87%) located in the outer membrane of
cells. Under laser excitation of dye-loaded cells suspended in D>O buffer,
oxygen phosphorescence arose with à lifetime of 58+2 Iis [79]. Singlet
oxygen quenchers methionine and sodium azide suppressed this phospho-
rescence. The phosphorescence intensity in cell suspensions was 6.4 — 23.2%
of that in à solution î é Üå dye in DzO. It was concluded that singlet oxygen
was generated in cell membranes and then was partly deactivated inside
membranes or diff used into DzO. According to Phillips, the phosphorescence
yield in suspensions of Toluidine Blue-loaded cells of Porphyromonas
gingivalis was much higher than in Kanofsky's experiments with eosin-
loaded leukemia cell suspensions [78]. It was suggested that Toluidine Blue
was located nearer to the outer membrane surface than eosin.
Dye-loaded erythrocyte ghosts were the most interesting among the model
systems investigated. The first studies of this model were reported by Girotti
er al. [80 — 82]. As photosensitizers, the derivatives of hematoporphyrin and
eosin were used. The dyes bound to erythrocyte membranes were shown to
generate singlet oxygen far less intensively than the same dyes solubilized by
à detergent in DzO. Firey and Rodgers investigated in detail rabbit erythro-
cyte ghosts loaded with TPPS [83]. In the analysis of fl uorescence and the
triplet state of porphyrin, two components were detected, which probably
corresponded to free and albumin-bound porphyrin. Under laser irradiation
(532 mn) of an erythrocyte ghost suspension in D>O, à weak 'Oz phospho-
rescence was observed. The phosphorescence signal was detected when the
TPPS concentration inside the ghosts was 0.3 IiM. In this case, the lifetime of
the phosphorescence was 46 Iis. It was efficiently quenched by sodium aside.
The 'Oz quantum yield was 8%, whereas monomåï c TPPS in DzO is known
to have à 65% 'Oz quantum yield. When porphyrin concentration inside
ghosts was 6 mM, à very weak luminescence appeared. Probably, it was not
related to '0 z, since it was not suppressed by azide and was not oxygen-
dependent. It seems that at à low concentration of TPPS, the phosphorescence
corresponded to 'Oz located in D20 outside erythrocyte ghosts. When TPPS
concentration was high, '0 > was quenched by porphyri
n itself. The
quenching rate constant was estimated to be 2.5õ10~ Ì 's ' [83].
Kanofsky reported 'Oz phosphorescence photosensitized by 5-(N-hexa-
decanoyl) aminoeosin bound to the erythrocyte ghost membranes suspended
in D2O [84]. Phosphorescence lifetime was about 23 iis. Since phospho-
rescence was suppressed by sodium azide and by substituting D>O for Í 20 ,
the author assumed that singlet oxygen responsible for phosphorescence was
located outside erythrocyte membranes. The same conclusion was made
when suspensions of erythrocyte ghosts containing pheophorbide à were
investigated [85].
Thus, all researchers, except Parker, observed phosphorescence of singlet
oxygen located extracellularly, even in those cases when photosensitizers
PH O SPH O R E SC EN C E ST U D IE S O F SIN G L ET O X Y G EN 679
w er e lo c at ed w it h in th e c el ls. T h e p h o sp h o re scen c e y i eld i n c e l l su sp en si o n s
w a s m u c h l ess a s c o m p are d to p h o to se n sit iz e r so lu t io n s . T h e d ete c ted ' O z
p h o sp h o re sc en c e w as su g g ested t o co r r esp o n d to à m in o r f r ac t io n o f si n g let
o x y g en m o lec u le s w h ic h su c c ee d ed in d if f u si n g o ut o f t h e c e l l s. M o st ' 0 2
m o l ec u le s a re l ik e ly t o b e d eac t iv at ed in sid e c e l ls d u e t o ef f i c ie n t ' 0 2
q u en c h in g b y b io m o l e c u les .
7 S i n g l e t O x y g e n Q u e n c h i n g b y B i o m o lec u le s
T h e rat e c o n st an t s o f ' 0 2 i n ter ac t io n w it h b i o m o lec u le s c an b e m ea su re d
u sin g c h em ic al ' O 2 tr ap s [ á , 86 , 8 7 ] o r b y q u en c h in g ' 0 2 p h o sp h o r esc en ce .
C h e m ic a l m et h o d s u su al ly p r o v i d e less a cc u rat e in f o r m at io n . E x p er i m en t a l
d ata o n ' O 2 p h o sp h o re sc en c e q u en c h i n g b y b i o m o lec u les ar e su m m ar iz e d in
T a b le 4 . A m o n g am in o a c id s, t h e m o st ef f i c ie nt are tr y p t o p h an , h i st id in e ,
m et h io n i n e an d c y ste in . T y r o sin e an d p h en y la lan in e ar e le ss e ff ic i en t b y a n
o r d er o f m ag n it u d e . O t h er am in o ac id s a re a lm o st in ac t iv e . T h e ac ti v it ie s o f
d ip e pt id e s an d tr ip e pt i d es w ere eq u a l t o t h e t o ta l a ct iv it ie s î Ã th e c o n st it u en t
am in o ac i d s [9 1 — 9 4 ] . M ath eso n et a l . p er f o r m ed th e f ir st m ea su re m en t s o f
t h e rate c o n st an t s f o r ' 0 2 in t er act io n w it h p r ot ei n m a cr o m o l ec u les [ 8 7 ] .
A c c o rd in g t o M at h eso n , th e ac t i v it ie s o f p r ot ein s c o in c id e d w it h t h e a ct iv ity
o f f r ee c o n st itu en t a m in o ac id s in so lu t io n . O u r ex p er im e nt s w it h ser u m
T a ble 4 . R ate co n stan ts î Ãsing let ox y gen d eactiv at io n by am ino ac ids and protein s in
D 2O , p D 7 — 8, m easured by the quench ing o f ' O ~ pho spho rescenc e.
Q u e n c h e r É~, M i s i R e f e r e n c e
T ry pt op han
H i sti d ine
M eth io n i ne
T y ro sine
C y stein
Pheny lalanin e
13-A lan ine
G ly c in e
B o v ine serum album in
H um an serum alb um in
Í à ï àë ser um alb um in
M el itt in
N euro peptide Y
In sul in
A n ser ine
C ar no sin e (3 .2 — 5 .6)õ 10
(4 — 4 .6 )õ 10
( 1.3+ 0 .1)õ 1 0 ~
(2 - 5 )õ 106
(0 .9 — 5 )õ 10
7õ 1()5
( Çõ 10
( 10'
(2 .6+ 0 .9 )õ 10
( 1.7Þ .5)õ 10 3
( 7.8+ 0 .3 )õ 10
5õ 10~
4 .5õ 10
7.5õ 10
Çx 10
Çõ 10 ~ [8 8, 89 , 9 3]
[89 , 9 1, 9 2, 93 ]
[90 , 9 3]
[9 3]
[90 , 9 3]
[93 ]
[92 ]
[93 ]
[8 8]
[88]
[9 5]
[94 ]
[94 ]
[94 ]
[9 1, 92 ]
[9 1, 92 ]
6 8 0
À . À . K RA SN OV SK Y, 1ã.
a l b u m i n s ( m o l e c u l a r m a s s , a b o u t 7 0 k D a ) s h o w e d t h a t t h e y w e r e 1 0 t i m e s
l e s s a c t i v e t h a n c o n s t i t u e n t a m i n o a c i d s [ 3 2 , 8 8 ] . K a n o f s k y r e p o r t e d t h a t
h u m a n s e r u m a l b u m i n q u e n c h e d ' 0 > f o u r t i m e s s t r o n g e r t h a n i n o u r m e a s u r e -
m e n t s , h e n c e , i t i s 2 . 5 t i m e s l e s s a c t i v e t h a n t h e c o n s t i t u e n t a m i n o a c i d s . A f t e r
d e n a t u r a t i o n b y à d e t e r g e n t ( l a u r y l s u l f a t e ) , t h e a l b u m i n a c t i v i t y d e c r e a s e d b y
à f a c t o r o f 2 [ 9 5 ] . A s f o r p r o t e i n s w i t h m o l e c u l a r m a s s e s o f 3 — 6 k D a ( i n s u l i n ,
m e l i t t i n , n e u r o p e p t i d e Y ) , t h e i r q u e n c h i n g w a s 0 . 7 — 0 . 8 3 o f t h e t o t a l a c t i v i t y
o f f r e e a m i n o a c i d s . A f t e r d e n a t u r a t i o n , t h e p r o t e i n a c t i v i t i e s i n c r e a s e d u p t o
t h e t o t a l a m i n o a c i d a c t i v i t y [ 9 3 , 9 4 ] . T h u s , d a t a o f d i ff e r e n t r e s e a r c h e r s
i n d i c a t e t h a t i n p r o t e i n m a c r o m o l e c u l e , s o m e a m i n o a c i d s a r e n o t a v a i l a b l e
f o r t h e ' 0 2 a t t a c k .
T a b l e 5 . R a t e c o n s t a n t s o f s i n g l e t o x y g e n q u e n c h i n g b y n u c l e o t i d e s , s a c c h a r i d e s a n d
o r g a n i c a c i d s i n D 2 O , p D 7 — 8 , o b t a i n e d b y m e a s u r i n g O ~ p h o s p h o r e s c e n c e
1
q u e n c h i n g .
Q u e n c h e r k , Ì i s 1 R e f e r en c e
W a t e r
T h y m i d i n e
4 - T h i o u r i d i n e
G u a n o s i n e
A M P
A T P
G l u c o s e
S a c c h a r o s e
O x a l i c a c i d
M a l o n i c a c i d
A s c o r b i c a c i d 4 õ 1 0 4 ( 3 + 1 ) õ 1 0 ( 1 . 2 + 0 . 2 ) x 1 0 s ( 5 + 1 ) õ 1 0 6 ( 4 . 3 Û . 5 ) õ 1 0 4 ( 4 ~ 1 ) 1 0 4 1 . 4 õ 1 0 4
1 0 4 ( 2 + 1 ) õ 1 0 4 ( 4 + 2 ) õ 1 0 4 ( 0 . 5 — 1 6 ) x 1 0 7 [ 3 1 , 9 5 ] [ 8 9 ] [ 8 9 ] [ 8 9 , 9 6 ] [ 8 9 ] [ 8 8 ] [ 8 8 ] [ 8 8 ] [ 8 8 ] [ 8 8 ] [ 8 8 , 8 9 , 9 7 ]
Î á æ å ã u n i v e r s a l c e l l c o m p o n e n t s a r e l i p i d s , c a r b o h y d r a t e s , n u c l e o t i d e s ,
÷ à ï î ì a n t i o x i d a n t s , p i g m e n t s . T h e r a t e c o n s t a n t s o f t 0 2 q u e n c h i n g b y s o m e
o f t h o s e c o m p o u n d s a r e p r e s e n t e d i n T a b l e s 5 a n d 6 . L i p i d s a r e f a r l e s s
e f f i c i e n t q u e n c h e r s o f s i n g l e t o x y g e n t h a n p r o t e i n s . T h e a c t i v i t i e s o f
p h o s p h o l i p i d s w e r e s h o w n t o b e e q u a l t o t h e t o t a l a c t i v i t y o f t h e c o n s t i t u e n t
f a t t y a c i d s . A m o n g n u c l e o t i d e s , g u a n o s i n e a n d t h i o u r i d i n e a r e t h e m o s t a c t i v e
w h e r e a s î ë å ã n u c l e o t i d e s w e a k l y i n t e r a c t w i t h s i n g l e t o x y g e n . O f î á æ å ã
c o m p o u n d s , u n i v e r s a l a n t i o x i d a n t s — a s c o r b i c a c i d , t o c o p h e r o l s , c a r o t e n o i d s
— a r e s t r o n g ' 0 2 q u e n c h e r s ( T a b l e s 5 a n d 6 ) . I t s h o u l d b e n o t e d t h a t c h l o r o -
p h y l l s , b a c t e r i o c h l o r o p h y l l s a n d p o r p h y r i n s c o m b i n e a n a b i l i t y t o g e n e r a t e
a n d q u e n c h ' 0 2 [ 2 3 , 1 0 2 — 1 0 4 ] . S i n g l e t o x y g e n q u e n c h i n g b y c h l o r o p h y l l s
a n d b a c t e r i o c h l o r o p h y l l s i s p h y s i c a l , i . å . , t h e y d e a c t i v a t e ' 0 2 m a i n l y w i t h o u t
d e s t r u c t i o n ; t h e r e f o r e , t h e s e p i g m e n t s s e r v e a s p r o t e c t o r s o f t h e p h o t o -
s y n t h e t i c m e m b r a n e s a g a i n s t s e l f - g e n e r a t e d s i n g l e t o x y g e n ( T a b l e 6 ) .
PH OSPHORESCEN CE STUD IES OF SIN GL ET OX Y GEN 681
T a b l e 6 . R a t e c o n s t a n t s o f s i n g l e t o x y g e n d e a c t i v a t i o n b y b i o m o l e c u l e s , o b t a i n e d b y
m e a s u r i n g 0 2 p h o s p h o r e s c e n c e q u e n c h i n g i n Ñ Ñ 1 Û .
Q u e n c h e r /ñ , Ì ÿ R e f e r en c e
W a t e r
E g g p h o s p h a t i d y l c h o l i n e
R e t i n a p h o s p h a t i d y l -
c h o l i n e
R e t i n a p h o s p h a t i d y l
e t h a n o l a m i n e
D i m y r i s t o y l
1 - Õ - p h o p h a t i d y l c h o l i n e
] 3 - C a r o t e n e
Z e a x a n t h i n
à - T o c o p h e r o l
R e t i n a l
C h l o r o p h y l l à
B a c t e r i o c h 1 o r o p h y l l à
B a c t e r i o c h l o r o p h y l l Ü
M e s o p o r p h y r i n I X
P r o t o p o r p h y r i n I X 2 . 8 õ 1 0 3 1 0 ) 1 0 4 1 0 ' 1 0 ' 1 ó 1 0 4 ~ ( 7 — 8 ) õ 1 0 9 7 x 1 0 ~ ( 1 — 7 ) õ 1 0 ~ 3 . 7 õ 1 0 ( 1 - 7 ) õ 1 0 ~ 1 0 ' 1 . 5 õ 1 0 9 2 õ 1 0 ~ 5 õ 1 0 5 [ 3 1 , 5 0 ] [ 9 8 , 9 9 ] [ 9 8 ] [ 9 8 ] [ 1 0 0 ] [ 2 1 , 2 3 , 9 8 , 1 0 1 ] [ 1 0 1 ] [ 2 3 , 3 1 , 9 8 a n d r e f s t h e r e i n ] [ 9 8 ] [ 2 3 , 1 0 2 - 1 0 4 ] [ 2 3 , 1 0 2 - 1 0 4 ] [ 1 0 3 , 1 0 4 ] [ 1 0 2 ] [ 1 0 2 ]
~M easured in. D2O
8 E stim ation of ' Î 2 L ifet im e in B iologica l Sy stem s
M a t h e s o n n o t i c e d t h a t t h e c o n c e n t r a t i o n s o f a m t n o a c i d s h i s t i d i n e , t r y p t o p h a n
a n d m e t h i o n i n e i n à l i v i n g c e l l a r e a b o u t 1 0 2 Ì . T h u s , u s i n g t h e
S t e r n — V o l m e r e q u a t i o n a n d t h e r a t e c o n s t a n t s o f ' 0 2 q u e n c h i n g b y t h e s e
a m i n o a c i d s , o n e c a n c a l c u l a t e t h a t x z i n c e l l s i s l e s s t h a n 1 I t s [ 8 7 ] . T h i s
c o n c l u s i o n w a s s u p p o r t e d Ú ó Ì î à ï [ 1 0 5 ] a n d l a t e r i n à s e r i e s o f o t h e r p a p e r s
[ 1 0 6 — 1 0 8 ] . W e n o t e d t h a t à c o m b i n a t i o n o f e q s . ( 3 . 3 ) ( t h e S t e r n — V o l m e r
e q u a t i o n ) a n d ( 3 . 4 ) y i e l d e d t h e f o l l o w i n g s i m p l e f o r m u l a f o r ~ ~ i n m u l t i -
c o m p o n e n t s o l u t i o n s :
òà = 1/Õ(l,, Ñ, ), ( 8 . 1 )
w h e r e / ~ , a r e r a t e c o n s t a n t s a n d Ñ , a r e m o l a r c o n c e n t r a t i o n s o f t h e c o m p o -
n e n t s T o a p p l y t h i s f o r m u l a t o l i v i n g c e l l s , î ï å n e e d s t o k n o w t h e ~ , a n d Ñ ,
v a l u e s f o r t h e m o s t a c t i v e c e l l c o m p o n e n t s . A s à r e s u l t , o n e c a n e s t i m a t e ò ~
a n d r e v e a l t h e c o n t r i b u t i o n o f i n d i v i d u a l c o m p o n e n t s i n t o o v e r a l l s i n g l e t
o x y g e n q u e n c h i n g b y t h e s y s t e m . T h u s , e q . ( 8 . 1 ) c a n b e c o n s i d e r e d a n
e x t e n s i o n o f t h e M a t h e s o n — M o a n m e t h o d w h i c h p r o v i d e s à u n i v e r s a l
682
À. À. KRA SNOVSKY, 1ã.
approach to the estimation of '0 > lifetime in living cells without the recourse
to time-resolved '0 2 studies. This method and the results of its application
were reported Úó this author in 1989 at the European Photobiology Congress
(Budapest) and at the Annual Meeting of the American Society for Photo-
biology (Boston) and published in [8, 31, 32, 106, 107]. Since 1990,
Kanofsky started to develop à related approach based on experimental '0 2
quenching by homogenates î È ûï éå8ãà1åä cells in heavy water [60, 78, 84,
95, 100, 111]. In fact, both methods were based on the same assumption that
'0 2 deactivation in living ñå11à |ç similar to that in à multicomponent solution.
Below, the data of both methods are compared.
Â!î î Ûð1àõò à. À biological system which is the most similar to the above
model is blood plasma. Estimation of t in this system was reported in
parallel papers [32, 95]. We used formula (8.1), concentrations of the main
quenchers of singlet oxygen presented in [109] and rounded À values from
Table 4 and literature data [32]. The À value for serum albumin corresponded
to î÷àã data [88]. For globulins the À values àãå not known, however, the
molecular masses of X~- and ~3-globulins are approximately equal to that of
albumin, therefore, we assumed that À values of these proteins were equal.
For î ë åã globulins, À,„ values were increased proport ionally to their mole-
ñè1àã masses. Calculations showed that the main 10 2 quenchers in blood
plasma are water and albumins. Water contributes 39% and proteins 50% into
overall quenching. The ~ä value was estimated to be 2.5 Itâ.
Kanofsky reported that the dependence 1/òä on plasma concentration (in
volume percent) is up to 15% à linear function of plasma concentration.
Extrapolation of the straight line to 100% plasma yielded ñä =1.04+0.03 Its
where contributions of water and proteins are 23 and 85%, respectively [95].
However, according to Kanofsky, albumin and water quenched singlet
oxygen stronger than in our experiments, and uric acid (not shown in Table
7) was found to be an effective '0 2 quencher [95]. If one introduces the
Kanofsky's À values for albumin (7.8õ103 Ì 's '), water (4.2õ103 Ì 's ')
and uric acid (3.6xIO~Ì 's ') into Table 7 and eq. (8.1), t = 0.9 1ts will be
obtained. Thus, both methods yield similar results.
Cell cytoplasm. It known that the cytoplasm of à plant and animal cell
consists of water (75 — 85%), protein with à mea
n molecular mass of 36 kDa
(10 — 20%), lipids with à mean molecular mass 0.7 kDa (2 — 3%); and îáæåã
compounds (2%) [110]. An average molecular mass of one amino acid is
about 120, therefore, main cytoplasm protein probably contains 300 amino
acid residues. It is reasonable to suggest that the contents of each of the 20
known amino acids in à protein macromolecule are equal. As only four amino
acids (tryptophan, histidine, methionine and cysteine) actively interact with
singlet oxygen, we can conclude that à protein macromolecule contains 60
residues of active amino acids. The cytoplasm density is known to be about
1 ô ñò ~. Hence, molar concentrations î Ãwater, protein and 1|ðÛû ï the cyto-
PH O SPH O R E SC EN C E ÁÒ Ø ) 1Å $ O F SIN G L E T O X Y G EN 6 83
Ta b le 7. Q uench in g of sing let ox y gen by hu m an b loo d p lasm a .
C o m p o n e n t s ~
Ñ~, ð Ì é~, Ì ts 1 À~Ñ õ 10 ~, s 1
W a t e r
S e r u m a l b u m i n
1 1, 1 - G l o b u l i n s
13 - G l o b u l i n s
Õ ~ - G l o b u l i n s
ó - G l o b u l i n
C a r b o h y d r a t e s ( p e n t o s e s a n d
h e x o s e s )
L i p i d s
F r e e t r y p t o p h a n
F r e e m e t h i o n i n e
F r e e h i s t i d i n e
B i l i r u b i n
A s c o r b i c a c i d
à - T o c o p h e r o l 5 x 1 0 7 6 0 0 1 0 0 1 3 0 2 0 7 1 0 0 2 0 0 0 1 0 0 4 0 1 5 0 1 0 1 5 0 2 0 Ç õ 1 0 4 2 x 1 0 s 2 x 1 0 s 2 x l p ~
1 0 ' 5 õ 1 0 â 1 0 4 1 ( ) ~ 4 õ 1 0 1 p 7 4 õ 1 0 7 2 õ 1 0 9 1 0 Ç õ 1 0 1 . 5 1 . 2 0 . 2 0 . 2 6 0 . 2 0 . 0 3 5 0 . 0 0 2 0 . 0 2 0 . 0 4 0 . 0 4 0 . 0 6 0 . 2 0 . 0 1 5
0 . 0 6
Ò î Ì à ! r a t e o f O ~ d e a c t i v a t i o n b y a l l c o m p o n e n t s , Õ ( é , Ñ , ) = 3 . 8 õ 1 0 â 1 ; O ~ È å -
t i m e , ~ ä = 1 / Z ( k q C q ) = 2 . 5 ð ü .
p l a s m a r e 4 4 , 0 . 0 0 4 a n d 0 . 0 4 Ì , r e s p e c t i v e l y . C o n c e n t r a t i o n s o f t h e m o s t
a c t i v e a m i n o a c i d s a r e a p p a r e n t l y h i g h e r b y à f a c t o r î 1 6 0 t h a n t h a t o f ð ã î t å m .
T h e m e a n k v a l u e s f o r a c t i v e a m i n o a c i d s i s a b o u t 2 õ 1 0 ~ Ì ' s ' ( T a b l e 4 ) .
1
T h u s , t h e t o t a l r a t e o f 0 2 d e a c t i v a t i o n c a n b e c a l c u l a t e d a s f o l l o w s :
E (k gC g) = 4 4 õ Çõ 10 + 0 . + 0 .0 4 x l p = 5 õ 10 â- ' .
Í å ï ñ å , ò ä = 2 0 0 n s w h i c h r e s e m b l e s t h e r e s u l t s o f p r i o r e s t i m a t e s [ 3 2 , 8 7 ,
1 0 5 — 1 0 8 ] . B e s i d e s , w e c a n s e e t h a t t h e q u e n c h i n g a c t i v i t i e s î Ã ð ã î 1 å 1 ï a m i n o
a c i d s g r e a t l y e x c e e d t h o s e o f l i p i d s a n d w a t e r . W a t e r c o n t r i b u t e s 2 — 3 % i n t o
o v e r a l l q u e n c h i n g .
R e s u l t s o f t h i s c a l c u l a t i o n a r e c o n s i s t e n t w i t h K a n o f s k y ' s s t u d i e s o f ' 0 2
q u e n c h i n g b y d i s i n t e g r a t e d l e u k e m i a L - 1 2 1 0 c e l l s . T h e d e p e n d e n c e o f t h e
i n v e r s e s i n g l e t o x y g e n l i f e t i m e i n D 2 O u p o n t h e c e l l c o n c e n t r a t i o n a p p e a r e d
t o b e l i n e a r . E x t r a p o l a t i o n î Ã t h e l i n e a r d e p e n d e n c e t o t h e 1 0 0 % c e l l c o n c e n -
t r a t i o n a l l o w e d à c o n c l u s i o n t h a t p r o t e i n s a r e m a j o r ' 0 > q u e n c h e r s , t h e c o n t r i -
b u t i o n î Ã w a t e r i s = 7 % , a n d t t , i n n o r m a l c e l l s s h o u l d b e 1 7 0 — 3 2 0 n s [ 1 1 1 ] .
E r y t h r o c y t e g h o s t s a r e k n o w n t o c o n t a i n p r o t e i n s a n d l i p i d s , w h o s e c o n c e n -
t r a t i o n s w i t h i n t h e g h o s t m e m b r a n e i s 6 0 0 — 8 0 0 a n d 2 0 0 — 4 0 0 m g m l [ 1 0 8 ] .
I f o n e a c c e p t t h a t t h e a v e r a g e m o l e c u l a r m a s s e s o f p r o t e i n s a n d l i p i d s a r e t h e
684 À . À . K RA SN OV SK Y, ,1ã.
same as in the cytoplasm, the molar concentrations of lipids and proteins
should be 0.4 and 0.02 Ì , which is much higher than in the cytoplasm.
Application of eq. (8.1) yields that the total rate of '0 > quenching by ghost
membranes is:
Õ(l,, Ñ, ) = 0.02õ60õ2õ10" + 0.4õ 105 = 2.4õ107 â ',
which leads to òä = 40 ns.
Kanofsky dispersed unsealed red cell ghosts in D2O by detergents SLS or
ÑÒÀÂ and studied the quenching of dye-photosensitized '0 > phospho-
rescence by this dispersion. Photosensitizers were eosin, Rose Bengal and
water-soluble tetraphenylporphin derivatives. Extrapolation of the results to
" 100%" ghost concentration led to à ~ä value equal to 24 — 130 ns in ghost
membranes [84] which is close to above estimate.
Retinal rods and chloroplasts. Detailed analysis of òä in outer segments of
retinal rods and chloroplast thylakoids is given elsewhere [8, 32, 106, 107]. It
was shown using eq. (8.1) that in outer segments of retinal rods the maj or '0 ~
quencher is rhodopsin and in thylakoids, carotenoids and chlorophyll. It was
found that the òä values are 70 ns in thylakoids and 400 ns in outer segments.
9 Estim ation of 'Oz Diff usion L ength
Òî understand the actual '0 ~ pathways in heterogeneous structures of the
cell, one should have information on the diff usion length of '0 > molecules
(1ä) i.å., the distance that singlet oxygen passes during its lifetime. According
to [112]:
1ä = „/áé ò (9 .1)
where D is the '0 ~ diff usion coeff icient. We proposed that D values are the
same for '0 > and ground-state molecular oxygen and equal to those reported
in [113] (Table 8). The D value in the lipid membrane is taken from [100].
The viscosity of blood plasma known to be 5 times higher [109]; the visco-
sity of cytoplasm, 10 — 100 times higher [110]; and that of thylakoids, 25
times higher [8] than that of water. Respectively, the diffusion coeffi ci ent is
lower by the same factors than in water. To estimate l~ (Table 8) we proposed
that in the cytoplasm, outer rod segments, erythrocyte membrane and
chloroplasts, the coefficient D is 30 times less than in water. It follows from
Table 8 that if singlet oxygen is generated in water or in lipid membrane, it
can diff use to à considerable distance &om the place of generation. In living
cells the diff usion length is much shorter being similar to or less than the
PH O SPH O R E SC EN C E ST U D IE S O F SIN G L E T O X Y G EN 68 5
Ò à Û å 8 . D i ff u s i o n l e n g t h a n d q u a n t u m y i e l d s o f s i n g l e t o x y g e n p h o s p h o r e s c e n c e i n
b i o l o g i c a l s y s t e m s .
M e d i u m
x a I t s D , c m s i a , A ã - ~
Ú â ~ðã ~ä ~õã
Í ã Î
Ð ã Î
L i p i d m e m b r a n e
B l o o d p l a s m a
C y t o p l a s m
E r y t h r o c y t e m e m b r a n e
R e t i n a l r o d o u t e r s e g m e n t s
C h l o r o p l a s t t h y l a k o i d s 3 . 1 6 8 7 1 0 . 2 0 . 0 5 0 . 4
0 . 0 7 2 õ 1 0 5
2 õ l 0 ~
1 . 2 õ l 0 ~
4 õ 1 0 6
7 õ 1 0 7
7 õ 1 0 7
7 x I 0 7
7 õ 1 0 7 1 9 0 0 9 0 0 0 2 2 0 0 5 0 0 9 0 4 5 1 3 0
5 5 4 õ l 0 7
8 õ 1 0 6
1 . 7 õ 1 0 6
1 0 - 7
5 õ 1 0 â
1 . 7 õ 1 0 à
l 0 — 7
l . 7 ~ 1 0 - â
m e m b r a n e t h i c k n e s s , t h e r e f o r e , s i n g l e t o x y g e n i s d e a c t i v a t e d m o s t l y i n c l o s e
v i c i n i t y o f t h e p l a c e o f g e n e r a t i o n [ 1 1 2 , 1 1 4 ] . H e n c e , w h e n p r o d u c e d i n s i d e
c e l l m e m b r a n e s s i n g l e t o x y g e n i s m o s t l y q u e n c h e d b y c e l l c o m p o n e n t s a n d
o n l y à m i n o r f r a c t i o n p e n e t r a t e s i n t o e x t e r n a l a q u e o u s m e d i a .
10 E st i m a t i o n o f 1O ã P h o sp h o r esc en c e Q u a n t u m Y i el d i n vi vo
T h e ~ ä v a l u e s c a l c u l a t e d a b o v e a n d e q . ( 5 . 1 ) a l l o w o n e t o e s t i m a t e t h e ' Î ã
p h o s p h o r e s c e n c e q u a n t u m y i e l d s ( ó « a n d ~ ð , ) i n t h e b i o l o g i c a l s t r u c t u r e s .
S i n c e ' Î ã i s m o r e r e a d i l y d i s s o l v e d i n h y d r o p h o b i c c o m p o n e n t s , w h e r e t h e
r a d i a t i v e l i f e t i m e i s a p p r o x i m a t e l y t h e â à ò å a s i n o r g a n i c m e d i a , w e a s s u m e d
t h a t i n v i v o x , = 4 s , i . e . t w i c e l e s s t h a n i n w a t e r . I t i s s e e n f r o m T a b l e 8 , t h a t
i n c e l l s o f l i v i n g o r g a n i s m s q , v a r i e s f r o m 1 0 7 t o 1 0 s . H o w e v e r , a c c o r d i n g
t o ( 5 . 1 ) , t h e q u a n t u m y i e l d o f ð h o t o s å n s t t t z å d p h o s p h o r e s c e n c e ( ó « ) d e p e n d s
o n b o t h < p , a n d t p a , w h e r e y a i s t h e q u a n t u m y i e l d o f s i n g l e t o x y g e n g e n e r a t i o n
b y à p h o t o s e n s i t i z e r . T h e l a t t e r s t r o n g l y d e p e n d s o n t h e s t a t e o f t h e p h o t o -
s e n s i t i z e r i n c e l l s a n d o n o x y g e n c o n c e n t r a t i o n . I t i s k n o w n t h a t t h e p h o t o -
s e n s i t i z e r s m o s t f r e q u e n t l y u s e d i n m e d i c i n e , p o r p h y r i n s a n d p h t h a l o -
c y a n i n e s , e ff i c i e n t l y g e n e r a t e s i n g l e t o x y g e n w h e n t h e y a r e m o n o m e r i c . I n
t h i s c a s e , t h e i r ~ ð ä v a l u e s ÷ à õ ó w i t h i n 3 0 — 8 0 % i n a i r - s a t u r a t e d s o l u t i o n s .
A g g r e g a t i o n o f p h o t o s e n s i t i z e r m o l e c u l e s l e a d s t o à s t r o n g r e r e d u c t i o n ,
s o m e t i m e s t o z e r o [ 3 1 , 3 2 , 5 9 a n d r e f s . t h e r e i n ] . O x y g e n c o n c e n t r a t i o n i n
c e l l s a n d t i s s u e s ( e x c e p t c h l o r o p l a s t - c o n t a i n i n g c e l l s ) i s m a r k e d l y d e c r e a s e d
d u e t o r e s p i r a t i o n [ 1 1 5 ] , w h i c h m i g h t b e à r e a s o n f o r a n a d d i t i o n a l d e c r e a s e
i n ~ ð ä . À s i m i l a r e ff e c t i s t h e c a s e i f s o m e p h o t o s e n s i t i z e r m o l e c u l e s a r e
a d s o r b e d o n p r o t e i n l o c i n o t e a s i l y a c c e s s i b l e f o r o x y g e n [ 1 1 5 ] . A n e s t i m a t e
p e r f o r m e d i n a i r - s a t u r a t e d m a l i g n a n t t u m o r s l o a d e d w i t h h e m a t o p o r p h y r i n
d e r i v a t i v e s y i e l d e d ä ä = 1 6 % [ 1 6 ] .
I n t h e p h o t o s y n t h e t i c a p p a r a t u s , ' 0 2 g e n e r a t i o n b y c h l o r o p h y
l l a n d
6 86 À . À . K RA SNOV SK Y, Jr.
bacteriochlorophyll is strongly decreased due to quenching of the pigment
triplet states by carotenoids. A s shown in [8, 106], in chloroplasts qi = 0.1% .
Thus, the phosphorescence quantum yield in the 4óå-loaded cytoplasm
should Úå less é àï 10 ~; and that in chloroplasts, about 10 " . Our set-ups
w ith time-resolved photon counting and laser excitation allow measurements
of '0 2 phosphorescence with the quantum yield of about 10 ~. Recently
W esseIs and Rodgers reported the detection of '0 2 phosphorescence w ith à
similar quantum yield on à set-up w ith stationary excitation and Fourier-
spectrometer [ 117]. Thus, according to the above estimates, phosphorescence
intensity i n vivo is below the threshold sensitivity of modern set-ups. This
agrees w ith the above described negative data on phosphorescence detection
in cells and tissues.
C o n c l u si o n s
Singlet oxygen phosphorescence is à reliable source î é ï Ãî ï ï à1|î ï about '0 2
generation and quenching by biologically significant compounds in organic
and aqueous media. A ttempts to measure '0 2 phosphorescence directly in
living cells have not been successful so far. However, the data obtained in
vitro allow one to j udge on the '0 2 lifetime, diff usion length, efficiency of
generation and deactivation pathways in biological systems. 1ï non-photo-
synthetic cells the main '0 2 quenchers are amino acids of proteins, in chloro-
plasts these are carotenoids and chlorophylls. They decrease the '0 > lifetime
to 40 — 400 ns, which corresponds to the diff usion length close to the thickness
of à membrane. Therefore, i n vivo '0 > molecules are deactivated in the
vicinity of hå sites î é Üå|ã generation. It is due to à short '0 > lifetime that the
phosphorescence quantum yield in biological systems is below the threshold
sensitivity î é ëå modern techniques. This is, apparently, the cause î Ë à|1èãåâ
in phosphorescence registration in vivo. Is it possible to create devices for
reliable measurements of '0 > phosphorescence in living tissues? This author
assumes it is possible, though, there are opposite opinions in the literature
[118]. The maj or limitation of modern equipment is à relatively low sensiti-
vity î Ãphotodetectors. M ore sophisticated photodetectors would, undoubted-
ly, instigate rapid progress in this field of research.
The work was supported by the Russian Foundation for Basic Research (grant
N o 98-03-32071à).
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