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