Vol. 64, No. 4, 1975
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
TRIPLET EXCITED STATES OF POLYCYCLIC AROMATIC COMPOUNDS AS PROBES OF THEIR MICROENVIRONMENT IN SERUM ALBUMIN COMPLEXES Nicholas E. Geacintov, John M. Ehosrofian Chemistry Department New York University,
Received
April
Thomas J. Flamer,
Thaddeus
Prusik
and
and Radiation and Solid State Laboratory New York, New York 10003, U.S.A.
lo,1975
Summary Using flash photolysis techniques, the triplet excited states of benzo(a)pyrene, pyrene, benz(a)anthracene and other aromatic hydrocarbons have been detected in complexes of bovine (and human) serum albumin dissolved in aqueous solutions at room temperature. The triplet lifetimes can be adjusted to any value within the microsecond-millisecond time domains by varying the partial pressure of oxygen from zero to one atmosphere, thus providing a useful probe on these time scales. Local oxygen concentrations as low as N 2 x LO-7M can be detected. In air saturated solutions, the triplet lifetimes are sensitive to pH dependent conformational changes of the host bovine serum albumin molecules.
INTRODUCTION Polycyclic human serum ing
is
aromatic albumin
hydrocarbons
(1)
and bovine
* 0.064
molecules/serum
dynamic
data
was concluded
phobic
region
binding
it
on the
and may be important ly toxic
with
ic effect
(4).
most
likely
The triplet
precursor
the
triplets
aqueous photolysis about
the
local
oxygen
concentrations
and are
sensitive
states it
The triplet ambient
of oxygen to conformational
(1).
complexed
and thus
type
and biologicalmolecules
in the photodynamare
albumin flash
probes
such
to monitor
of photooxidation,
of the host
of
information
to small
can be used
the rates
time
serum by the
give
the
degradation
the decay
bovine
which
of (3)
sensitizers
that
molecules
changes
This
aromatic
can be determined times
to a hydro-
photosensitized
with
of bindon thermo-
carcinogenesis
sensitizers
in the
conditions
Based
hydrophobic
is shown
of the aromatic under
molecule
with
The extent are bound
of these
decay
complexes
2).
Many polycyclic
are also states
molecules
accessibility
(1,
of these
at room temperature
technique.
as molecular
excited
communication
of aromatic
solution
(2).
to chemical
systems.
electronic
In this
albumin
the hydrocarbons
transport
or enzymes
to form
molecule
of the albumin
in living
proteins
of proteins.
that
in relation
in the
compounds
complexed
serum
albumin
surface
has been considered
are known
macromolecule.
in
Vol. 64, No. 4, 1975
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
EXPERIMENTAL Crystallized were
bovine
purchased
aromatic
from Calbiochem
hydrocarbons
crystallization
(Rutgers
were
prepared
to aqueous
agitating
Whatman No.1 hour.
paper
flash
state
state
Tl' lamp in our
may decay
intersystem
crossover
A monitoring
beam of light
experiments)
is
in time.
the triplet
lifetime
electrically
either
in the signal used,
T. with
averaging
a conventional
frequency 347.1
(347.1
The laser
Ml).
was used when the
dependent
system
were
of 0.5-1.0%
technique,
cyclohexane solution
(singlet are
shown
the same in both is slightly a lifetime composition
path
air
spectra decay
times
were
lifetime
z
saturated
path
S
2; 4th
plots
- 12 Volt
tungsten T
n As the
absorption
absorption
yields photo-
a multichannel
analyzer
photolysis
systems
(50 joules)
were
and a
(50-100
millijoules
length
of 10 cm
at
range,
and whereas
20 cm) was used when the Transient
determined for
= 47 nsec)
absorption
absorptions
in Fig.
changes are wavelength
1. by the single
benzo(a)pyrene and in air
the BSA solution
and degassed
triplet
triplets beam,
the microsecond
length
are shown
decay
in Fig.
system
of
(Sl-+So)
lowest
transient
system
the
(Sl)
was monitored
of flash
range.
Typical
to upper
or with
The transient
be detected.
and typical
a 100 Watt
the decay
was in
(sample
state
to the
the CT1 + So>, of this transient
has a sample
lifetime
excites
singlet
of the monitoring
laser
in the millisecond could
The fluorescence counting
system
of light
by fluorescence
So
lamp-pumped
run) ruby
flash
Tl
Two types
one-half
The effective
to
(from
work,
flash
triplet
the conventional lifetimes
mode.
rpm for
of 10 -6 -5 x 10-6M.
(SldTl)
an oscilloscope
xenon
doubled
time
In this
through
excited
used to excite
The decay
filtered
at 10,000
a strong
to the first Sl
hydrocarbon
procedures.
range
re-
albumin
and subsequently
were
by these
technique
(So)
by repeated
aromatic
centrifuged
resulting in an attenuation 'T,'T 1 1, triplets decay to the ground state decreases
purified
(HSA)
The
hydrocarbon-serum
ground
was in the
hydrocarbons.
pure.
(3mg protein/ml)
removed
photolysis
or by nonradiative
finely
and then were
singlets
the aromatic
were
The suspensions
of hydrocarbons
In the
A.G.)
solutions
12 hours.
filter
concentration
ground
Werke,
by adding
The microcrystals
and human serum albumin
The polycyclic
protein
at 5°C for
(BSA)
and are electrophoretically
from ethanol.
complexes crystals
serum albumin
solutions.
the
photon
in degassed saturated decay
curve
BSA was
In BSA the decay
nonexponential and can be decomposed into two decays, one with Using straightforward deof 17 nsec and the other 35 nsec. - 92% of the and integration techniques it can be shown that
1246
Vol. 64, No. 4, 1975
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
I
I
(A)
(El
c
o.sol--J I
l h, I
lrllllllrlrrllt’ 40
I 420
520
460
1
WAVELENGTH,
Fig..l(A).
emitted
correspond
may be one major
binding
The results obtained
with
1
I 460
I
I
1
I 500
I
I
IW 540
n m
Pyrene - spectral dependence of transient absorption; I, incident intensity, I transmitted intensity.-O-0 conventional flash, l . . . laser flash photolysis system. Benzo(a)pyrene. Complexes in bovine serum albumin in H20, pH 5.1, 24°C. In the laser experiment a dichroic mirror, which cuts off partially below 440 nm, was used as a beam splitter.
(B) -
photons
I
to the site
obtained BSA is
longer
lifetime
indicating
that
there
in BSA (2).
with
described
HSA and BSA were in detail
in
quite
this
similar
and the data
communication,
THE TRANSIENT ABSORPTION IS DDE TO TRIPLETS Photogenerated radicals
or triplets.
ionization decay
time
tion
as the
(5)
to triplets
be the spectrum
triplets.
is
transitions) same as that for
giving
pyrene
rise
spectra
the decay
at all
the (6)
time
wavelengths in Fig.
l(A)
1247
two-photon
which
may have a similar
possibilities transient
for
we performed absorption
assigning
of the transient exhibiting
of the phosphorescence shown
excitation,
of these
to show that One criterion
that
may be due to cations,
to cations
In view
experiments
due to triplets.
(Tn+Tl
absorption
In the case of laser
can occur
the appropriate indeed
transient
this
transient
characteristic
absorp-
absorption absorption
(T 1 -S o emission). is
is
of triplets
must The (6,7).
Vol. 64, No. 4, 1975
BIOCHEMICAL
IO
20
30
40
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
30
70
60
CHANNEL
Fig.
In the case of benzo(a)pyrene but
a maximum at not
pyrene tion cence
identified
time
cay time triplet
conclusively
(6).
cannot
yields.
100110
120
decay
(- 83 msec)
identical nature
within of the
with
130
time
be observed
degassed
because
error
excitation the laser
of benzo(a)-
of the both
decay
the characteristic
low concentra-
the
phosphores-
of benzo(a)pyrene
and transient spectrum
(see Fig.
Comparisons
spectra to triplets
polymethylmethacrylate
(- 80 msec)
transients.
absorption attributed
The phosphorescence
absorption
thick
experimental
spectra using low intensity and high photon flux using
transient
previously
We thereforemeasured
and transient
in a 20 micron
The phosphorescence
l(B)),
have been
in BSA complexes decay
(Fig.
- 470 nm
and low quantum
dissolved
are
SO
Decay of singlets (fluorescence decay) and triplets of benzo(a)pyrene. Sl*S,(CH)... decay of singlets in degassed cyclohexane solution, 1.81 nsec/channel. sl+so(~SA).... decay of singlets in BSA complexes, T +Tl (BSA).... decay of triplets in BSA complexes monitore 2 by flash photolysis. decay of triplets by flash photolysis in Tn + Tl(PMMA).... polymethyl methacrylate films. T1So.... decay of'triplets monitored via phosphorescence of the same polymethyl methacrylate film.
2.
with
60
NUMBER
(-
absorption shown
2) thus
of transient
10-2M)
(PMMA) film. in Fig.
confirming
del(B) the
absorption
with the xenon flash apparatus are shown in Fig. 1, and are similar
1248
Vol. 64, No. 4, 1975
within
BIOCHEMICAL
experimental
in both
error
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
indicating
that
triplets
are being
observed
cases.
QUENCHING BY OXYGEN In the presence we have
observed
of oxygen
that
(with
the triplet
the concentration
lifetime
7T
denoted
follows
by CO,]),
the Stern-Volmer
law
0
where
is the lifetime in rigorously degassed solution and $ is TT quenching constant. The solutions were degassed by 7-10 consecutive
the
freeze-pump-thaw
cycles.
benzo(a)pyrene value
of
benzene
rTo
N 80
that
hydrophobic
In air
with
BSA and for
hexane
(8,9)
carbons This
is
not
group methyl
fect.
While
surface
The relative fluorescence (11) *
are
decay
Pyrene
is
available (8)
KT
are for
fluid
It
appears in a rigid
gives
to 0 2
extent
an exception
in cycloI.
In
polycyclic
benzo(e)pyrene
5
hydro(benzo(a)-
exhibits
The presence
quenching
appears
to have
have
the
same
in shape
and
may
Benzo(a)pyrene that than
it
is
the highest
of the bulky
to a higher
constant little
fit
more coma lower
in a region
of the complexed
molecules
can also
of fluorescence
quenching.
Since
to detect because
measurable of its
1249
ef-
molecular
exhibits located
butyric
in the case of benzo(e)pyrene.
are in the nanosecond
required
constants
and thus
KT
in air
> KT (benzo(e)pyrene).
rise
of BSA.
and
complexed
shown in Table
energy
indicating
range
compounds
aromatic
(pyrene)
is more elongated
accessible
times
In degassed located
quenching
et al
and benzo(e)pyrene
accessibility
usually
the
yrCO,l >> (7,OP
several
the lowest. acid
the
approaches
in the microsecond
triplet
region
by determining
and for
msec
msec long. are
in benz(a)anthracene
benzo(e)pyrene less
0.30
since
in BSA since
the former
is
is
for
> l$
and pyrene
in a hydrophobic
% in BSA which
sures
increasing
benzo(a)pyrene
than
is (1)
solutions
substitution
area,
value
probed
(10)
in pyrenebutyric
while
rT
the
followed
constant
rTo
eq.
by Patterson with
20
PMMA films.
hydrocarbons
values
KT (benz(a)anthracene)
quenching
pactly
KT
comparison,
and solid
trend
acid
from
The
decreases
pyrene)>
hand,
solutions
as determined
fluid
the rigid
is
2) which
of the BSA molecules.
directly
solutions.
rTo
(see Fig.
aromatic
region
saturated
can be calculated saturated
in
the
in BSA
long
msec
on the other
results
and inert
66
obtained
msec
solution
from these
For pyrene
is
range,
high
fluorescence relatively
long
oxygen
be the pres-
quenching singlet
Vol. 64, No. 4, 1975
BIOCHEMICAL
Table
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
1.
Triplet lifetimes and oxygen quenching constants KT in air-saturated solutions of polycyclic hydrocarbon-BSA complexes in water pH = 5.1, ionic strength u = 0, 24°C. At 760 mm atmospheric pressure [02] = 2.76 x lO-4 moles/liter, calculated from a handbook value of Henry's law constant (15). The KT values in cyclohexane are from ref. (3).
Triplet sec(x benzo(a)pyrene benzo(e)pyrene pyrene pyrenebutyric
acid
benz(a)anthracene 7,12-dimethyl benz(a)anthracene
lifetime. is
In BSA complexes
338 nsec,
slightly of free
pyrene
of pyrene
is
quenching
Ks(H20)
= 9.8
fusion
controlled
ten
times
ments
for
which
is
tained
but
proceeds
22 f 1 23 i 1
1.7 1.6
f 6.10 k 0.1
19.3 ----
lit
value
(11). to
than is
note
When aqueous ence of
N 10w5M
presence
of this
due to quenching,
indicate
yield
Ks(BSA)
obtained
for
both
s/s
with
singlet
(8)
transfer
oxygen
molecule.
close
route
are
and increases
exposed
to light,
of oxygen to
1250
for
giving
less several
this
x 10'
ob-
the ratios
of triplets
is
about
(11). acid
the triplet
N 50 msec after
(12)
of 1.34
complexes
initially
is
measure-1 set
to the value
(9,lO)
of benzo(a)pyrene-BSA
concentration
pyrene
and Weber
when the quenching
energy
solutions
is
and
to the dif-
and Weber
the value
and pyrenebutyric This
The
Similar lit M-1
= 1.5 x 10'
by Vaughan
pyrene
6% 10.
that
in water.
7s
x 108,
close
Ts
also
quenching
law.
= 9.7 is
is
water
quenching
in BSA by Lakowicz
statistics
oxygen
results
in BSA than
agreement
Pyrene
In degassed
O2
lifetime
the fluorescence
the Stern-Volmer
the value
by the partial reactive
the singlet
solutions.
be determined.
in reasonable
constants by spin
02,
and thus
These
acid
that
of
pyrene are Ks(BSA) -1 -1 M set ; the latter value
9-vinylanthracene
of quenching
highly
16.8 ----
for
accessible
larger
We also predicted
f 0.05 f 0.10
can also
pyrenebutyric
for
0.95 1.7
in air-saturated
constants
x 10'
less
compound,
38 f 2 22 It 2
199 nsec and obeys
singlet
M-lse~-~(~lc~) 26.3 ----
(- lo-%I)
in water
lit
1.3 f 0.05 1.9 f 0.10
in the absence
in water
1(x10-8)
28 * 1 19 * 1
and is 310 nsec
soluble
KT(cyclohexane)
KT (=A) lit M-lsec-
Ligfetime 10 )
of 9 by oxygen
rise
to the
in the lifetime than
presin the
one msec
hours.
This
Vol. 64, No. 4, 1975
Fig.
3.
experiment
indicates faster
diffusion
it
oxygen
estimated
be detected
of
Quantitative rates
is O2
RESEARCH COMMUNICATIONS
by this
consumed
experiments
oxygen
T(sec) of pyrene aqueous solutions results are obTj
is
concentrations
from
only
of this
and local
in relative
units
in some photooxidation
by diffusion
in water
of consumption
that
being
can be replenished
coefficient
in obtaining is
that
than
at 24°C (13)). It
AND BIOPHYSICAL
0 . . . . pH dependence of triplet lifetimes in BSA complexes at 24°C in air saturated and at ionic strength P = 0.01. (Similar tained with 3,4 benzo(a)pyrene). -. . . D pH dependence of the viscosity (14).
process (the
BIOCHEMICAL
the atmosphere
N 2 x lo5
type
should
concentrations as low as
cm2 set-'
be useful of oxygen.
N 2
x
10m7H
can
pH 4.3
and
technique.
EFFECTS OF CONFORMATIONAL CHANGES BSA above
is
known
pH 10.5.
viscosity
Below
which
is
the BSA molecule is
These
vironment
is
are
The results
expansion is
a rather
to the increase in
attributed
in Fig.
sharp
increase
of the hydrodynamic
the triplet
hydrocarbon probes
below
of the BSA molecule lifetime
to an enhanced
summarized
as sensitive in protein
there
The expansion
of the aromatic
effects
be utilized
related
by a decrease
which
cessibility
a reversible
pH N 4.3
(14).
accompanied
solutions,
to undergo
to oxygen
of oxygen that
changes
the
and accomplex.
triplets
in their
local
complexes.
described
here
show that
1251
triplet
excited
of
pH 4.3
saturated
in the protein
3 and indicate
of structural
volume
below
in air
diffusion
in the
states
can
may en-
Vol. 64, No. 4, 1975
complement
BIOCHEMICAL
the results
obtained
long
lifetimes
which
time
domains
by varying
some important
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
with
fluorescent
can be adjusted
within
probes.
the oxygenconcentration,
advantages
over
the
commonly
Because
of their
the microsecond-millisecond the triplets
may offer
used fluorescent
probes.
ACKNOWLEDGEMENTS This Grant
investigation
CA 14980
from
was supported the National
Council
City
of New York
Grant
Atomic
Energy
Commission
grant
is
also
mission
acknowledged. to use his
Lakowicz
for
some
We are
by Public
Cancer
No. U-2312,
xenon
lamp flash
helpful
comments.
assistance
and Solid
to Professor photolysis
Service
Research
and by a Health
Partial
to the Radiation indebted
Health
Institute
D.I.
apparatus
Research from an
State
Laboratory
Schuster
for
and to Dr.
perJ.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 80 9. 10, 11. 12. 13. 14. 15.
Franke, R., (1968) Biochem. Biophys, Acta 160, 378-395. Biochem. Biophys. Acta, in press. Bothorel, R. and Desmazes, J.P., Franke, R., (1969) Mol. Pharmacol. 5, 640-657. Spikes, J.D. and MacKnight, M.L., (1970) Ann. N.Y. Acad. Sci. 171, 149-161. Fisher, M.M., Veyret, B. and Weiss, K., (1974) Chem. Phys. Lett. 28, 60-65. PhotoLabhart, H. and Heinzelmann, W., (1973) in Organic Molecular physics, edit, J.B. Birks, John Wiley and Sons, 297-355. Schomburg, H., Staerk, H. and Weller, A., (1973) Chem. Phys. Lett. 22, l-4. Patterson, L.K., Porter, G. and Topp, M.R., (1970) Chem. Phys. Lett. 7, 612-614. Gijzeman, O.L.J., Kaufman, F. and Porter, G., (1973). J, Chem. Sot. Farad. Trans. II 2, 708-720. Benson, R. and Geacintov, N.E., (1973) J. Chem. Phys. 59, 4428-4434. Lakowicz, J.R. and Weber, G., (1973) Biochemistry 12, 4161-4179. Vaughan, W.M. and Weber, G., (1970) Biochemistry 2, 464-473. St. Denis, C.E. and Fell, C.J.D., (1971) Can. J. Chem. Engnr. 49, 885. Tanford, C., Buzzell, J.G., Rands, D.G. and Swanson, S.A., (1955) J. Amer. Chem. Sot. 77 6421-6428. 37th Edition, Chemical Rubber Handbook of Chemistry and Physics, Company, p. 1606.
1252