Journal of Photochemistry

and Photobiology,

B: Biology, 5 (1990)

467 - 480

467

THE EFFECT OF THE ANTIOXIDANT SPERMINE ON THE PHOTOPHYSICAL REACTIONS OF TRYPTOPHAN IN AQUEOUS SOLUTION AT ‘77 K GHARIB S. MAHMOUDT and THOR B. MELQ Institute of Physics, AVH University of Trondheim, Trondheim (Norway) (Received September 8,1989;

7055 Dmgvoll,

accepted November 15,1989)

Keywords. Photo-oxidation, spermine, photolysis, UV effects.

summary Fluorescence, phosphorescence and electron paramagnetic resonance techniques were used to investigate the effect of the antioxidant spermine on the initial photophysical reactions of tryptophan (Trp) in aqueous salt solutions at 77 K. At low concentrations of Trp (3.5 X lo-’ M) a ground state complex was formed between one Trp and two spermine molecules (a 1:2 complex). Complexed Trp was photodegraded at a rate 65% lower than the free molecule due to a change in the charge-transfer character of the excited ‘L, state. At high concentrations of Trp (3.5 X 10m3 M) the phosphorescence was almost completely quenched due to hydrogen-bond formation between two neighbouring Trp molecules. A strong complex was formed between this Trp dimer and one spermine molecule on addition of spermine (a 2: 1 complex). Spermine enhanced intersystem crossing in one of the two Trp molecules in the 2:l complex and phosphorescence was observed. From this triplet state the tryptophyl radical was formed with high efficiency by hydrogen-atom transfer. The yield of radical formation from the triplet state in the 2:l complex was much larger than from the excited singlet state in the 1:2 complex.

1. Introduction The photochemistry of tryptophan (Trp) has been extensively studied [ 1 -121. Its main photo-oxidation product is N-formylkynurenine [lo]. The neutral Trp radical, which can be formed from an excited singlet or triplet state [lo], is an intermediate substance in the chain of reactions leading to the stable photo-oxidation product. At elevated exciting fluence rates TAutbor to whom correspondence loll-1344/90/$3.50

should be addressed. @ Elsevier Sequoia/Printed

in The Netherlands

triplet absorption can also occur [ 121. Other factors that complicate the photochemistry of Trp are the existence of different ground state rotamers, the acid-base properties of the amino and carboxyl groups and the involvement of the imino group of the indole ring in interactions with the solvent or other solute molecules [lo]. Charge-transfer complexes are also formed between Trp and neighbouring solvent molecules [ll]. The photophysics and photochemistry of Trp are quite well known despite their complexity. When the sensitizing action of a substance is to be explored, Trp may therefore be selected as a test molecule. The aim of this investigation is to see how the naturally occurring antioxidant spermine (Spr) [13 - 171 acts on the photochemical reactions initiated by light absorption in the Trp molecule. Neutral aqueous solutions of Trp were studied at 77 K in order to limit the number of reaction steps following the photoionization event. Salt was added to the solution to prevent the aggregation of Trp molecules on freezing [2].

2. Materials and Methods L-Tryptophan (Trp) was purchased from Sigma Chemical Company and spermine (Spr) from Aldrich. Fresh solutions of Trp and Spr in phosphate buffer were prepared before each experiment. The buffer was made by dissolving KH2P04 (1.36 g), Na2HP04 (1.78 g) and NaCl (7.6 g) in 1 1 of deionized water from a water purifier system (Milli R/Q, Millipore Corporation). Trp and Spr from stock solutions were mixed and diluted to the desired concentrations prior to each experiment. The sample was transferred to a quartz tube (inner diameter, 3 mm), which was inserted in a quartz Dewar (Bruker) filled with liquid nitrogen. The same sample arrangement was used both for the luminescence and the electron spin resonance (ESR) measurements. In the luminescence measurements the Dewar was clamped in an X-Y-Z holder attached to the sample house of the fluorometer. To prevent water condensation, pure nitrogen gas was passed around the tip of the Dewar. The luminescence spectra at 77 K were recorded using a Fluorolog 222 (Spex Industries) fluorometer, with the Datamate (the computer part of Fluorolog 222) linked to a VAX computer. The wavelength of the excitation light was 280 nm and the slit width was 1.5 mm for both the excitation and emission monochromators (bandwidth, 2.7 nm). The exciting fluence rate, measured with a calibrated thermopile detector (Sensors, Inc., type 15), was 0.2 mW cmw2. The ESR investigations were performed using a Bruker ER 1OOD spectrometer equipped with a signal averager (EC & G model 4202). An ABC 80 (Scandic Metric) computer was used to read and transmit data from the signal averager to the VAX computer. The radicals were formed on irradiation of the samples in the cavity of the ESR instrument at 77 K using a 900 W photoirradiator equipped with a monochromator (Applied Photo-

469

physics). The excitation wavelength was 290 nm and the fluence rate was 4 mW cm- 2. A UV filter was placed after the irradiator to absorb all quanta with wavelengths less than 280 nm.

3. Results 3.1. Luminescence measurements As the concentration of Trp is systematically increased, the fluorescence spectra of the frozen solutions are progressively shifted to the red and the phosphorescence spectra are substantially quenched (Fig. 1). At low concentrations both the fluorescence and phosphorescence peak intensities (Fig. 2) vary linearly with [Trp] . At the highest absorbances nearly all incident quanta are absorbed by the specimen and hence the fluorescence intensity If levels off at high concentrations. The ratio of the two luminescence intensities (Fig. 2) is constant at low concentrations and declines quickly at higher concentrations. At the highest Trp concentration (3.5 X 10e3 M) the phosphorescence spectrum is almost completely quenched. Furthermore, as the phosphorescence is quenched it can be seen that it consists of two independent spectra (see Fig. 1). The spacing between the two sets of phosphorescence emission lines is 10 nm, and the spectrum displaced to shorter wavelengths is more rapidly quenched, as [Trp] is increased, than the long-wavelength spectrum. Finally, both If and the phosphorescence intensity IP vary linearly with the exciting fluence rate. On the left-hand side of Fig. 3 the relative yields of fluorescence qf and phosphorescence q, are plotted us. [Spr] from samples with [Trp] = 3.5 X lo-’ M. nf, scaled to unity when

LEGEND --3.5 E-3 M a.75 E-4 M _____.__..__. 35 E-4 M a75 E-5 M _-_-.-____ 35 E-5M ..-..- .._.. _... 3.5 E-6 M 300

325

350

375

400

425

450

475

500

525

f

3

Wavelength , nm Fig. 1. Fluorescence and phosphorescence spectra of !l’rp in aqueous salt solutions of increasing concentration. For [Trp] = 8.75 X 10m4 M two different phosphorescence spectra are seen.

470

4=

c

\ .-

:

i

LEGEND

* Ratio -------. 0 Phos 0 Fluor

Fig. 2. Maximum fluorescence ratio as a function of [ Trp].

and phosphorescence

(at 432 nm) intensities and their

:j__*.‘#.;,.: 0

2

4

6

8lOP14161820

Eiprl , mM

L.........

‘02468OPl4BB20

&w1, mM

00;

02468OPUl68~

CsPrl, mM

Fig. 3. Relative yields of fluorescence and phosphorescence and the phosphorescence to fluorescence ratio as a function of [Spr]. On the left-hand side of the figure the concentration of Trp is 3.5 x 10ms M; in the middle it is 3.5 x 10m4 M and on the right-hand side it is 3.5 x 10e3 M.

471

[Spr] is zero, approaches an upper limiting value of 1.65 at high [Spr] values. Ip seems to reach a maximum at an intermediate [ Spr] value (8 mM), and the luminescence ratio decreases monotonically as a function of [ Spr]. For further treatment of the data (Fig. 4, upper) it is assumed that the fluorescing Trp entities exist in two populations, either as monomers with Q scaled to unity or complexes with Spr with a higher yield of 1.65 (asymptotic value). The ratio of complexed to non-complexed Trp molecules can be derived from Fig. 3 (left) and in Fig. 4 (upper) it can be seen that this ratio varies with [ Spr] *. In Fig. 5 the normalized luminescence spectra of Trp from solutions with [Trp] = 3.5 X 10e3 M and varying values of [ Spr] are shown. IP increases rapidly for the initial Spr additions and the fluorescence maximum is shifted to lower wavelength. Only one of the two highly quenched phosphorescence spectra increase in intensity when Spr is added. The short-wavelength spectrum remains quenched when Spr is added. On the right-hand side of Fig. 3, If, Ip and IJI, are plotted as a function of [Spr] for the high concentration case ([Trp] = 3.5 X low3 M). qf seems to decline slightly with the initial Spr additions (below 2 mM); however, there is a rapid initial increase in qP. This is not observed when [ Trp] = 3.5 X lo- ’ M. The relation between IP (the long-wavelength spectrum) and [Spr] is given further data treatment in Fig. 4 (lower). The ratio of IP to the difference between the maximum and actual phosphorescence (which gives the ratio of non-complexed to complexed Trp) is plotted for each [Spr] value. A linear relation is found with a slope of 2.0 X lo3 M-l. In the middle part of Fig. 3 the same measurements as above are shown for an intermediate Trp concentration (3.5 X lop4 M). If reaches an upper

[Sprl , mM Fig. 4. Upper: ratio of the number of Trp-Sprz complexes to the number of Trp monomers as a function of [Spr12. Lower: ratio of phosphorescent dimers (Trp-Spr) to nonphosphorescent dimers us. [Spr].

412

LEGEND SprilSmt.4 _______...._. SC%2.ord.l -------. SW 1.5 mM ----Spr 10 mM --SQro5mh4 ScfOOmM 0

Wavelength , nm Fig. 5. Luminescence spectra of solutions with [Trp] = 3.5 for [Spr].

x

lop3 M and various values

asymptotic value, as in the low concentration case, when [Spr] is increased. The upper limiting value is smaller and is reached more quickly in this case. In addition, in this case the long-wavelength phosphorescence spectrum is very sensitive to minor Spr additions (below 2 mM). At high concentrations of Spr (5 - 20 mM Spr) Ip and the luminescence ratio decline in the same way, regardless of [ Trp] . 3.2. Photodegradation measurements The fluorescence intensity If of solutions ([Trp] = 3.5 X lo-’ M) that are continuously and uniformly irradiated decreases exponentially with illumination time. In Fig. 6 the rate constant h, of this exponential decay is shown as a function of [Spr] in the sample prior to freezing and illumination. lz, decreases when [Spr] increases. This graph has an inverse relation to [Spr] compared with that of qf (for the same [Trp], see Fig. 3). The yield 7r of Trp photodegradation can be estimated from these measurements. Iz, of Trp, derived from the graph of If us. illumination time, is 8.0 X 10e5 s-l (no Spr added). This number is the probability per unit time for conversion of a Trp molecule into a photoproduct ((dN/dt)/N = -k, N is the instantaneous number of Trp molecules). Since the fluence rate I0 is known (see Section 2), the probability per unit time for a Trp molecule to absorb (k,) a quantum from the light beam can be estimated from the expression k, = (IO X E X 1000 cm3/NA), where E is the molar absorption coefficient at 280 nm (5500 cm-’ M-‘) and NA is Avogadro’s number. This gives k, = 2.58 X 10d3 quanta s-‘. Therefore the yield of Trp photodegradation, k,/k,, is 0.031. This value agrees with the values reported elsewhere in the

473

II) I I p

z 2

E (0

.i\

.6

\

i 1 I

\

’ 1

.6-

\ \

*; s 0

i\

ii, \

a,

.

z.4 u

%

\

\ \

\ \

‘i

LEGEND

\ \

‘d__ --x-__ 25 0

. 2

I 4

. 6

. 6

. 10

[Sprl

L

--. 12 ,

. 14

---* . 16

. 16

. 20

1

----x Trp 39-4 --* Trp3!X-5 2:2

mM

Fig. 6. Rate constant of the exponential photodegradation The rate constants are normalized to unity at [Spr] = 0.

of Trp as a function of [Spr].

literature [lo]. The values of lz, are also shown us. [Spr] for [Trp] = 3.5 X 10e4 M (Fig. 6). Minor Spr additions are needed in this case to cause a similar reduction in the rate constant. For the highest Trp concentration (3.5 X low3 M) the photodegradation can no longer be described by a single exponential decay. The peak If values plotted VS.the irradiation time for different Spr additions (Fig. 7) show that the effect of Spr on the photodegradation is largest at low concentrations (below 2 mM), which is also the case for the phosphorescence changes. The photodegradation of Trp can satisfactorily be described as a sum of two exponentially varying terms with different rate constants. The ratio of the rate constants is 40 f 5. The photoproduct is fluorescent [9] (Fig. 8) and Fig. 10 (left, see Section 3.3) shows that the relative intensity of its emission, after photoirradiation for 300 s, has the same dependence on [Spr] as the phosphorescence and radical yield. 3.3. ESR measurements The ESR spectra of photoirradiated frozen Trp solutions (3.5 X lop3 M) containing various concentrations of Spr (Fig. 9(A)) are mainly due to the neutral Trp’ radical [18 - 251. The size of the electron paramagnetic resonance (EPR) signal after photon-radiation increases with [ Spr] and in Fig. 10 the radical yield, measured as the peak-to-peak height of the ESR spectrum, is shown as a function of [Spr]. Below 1 mM the yield increases quickly, in the same way as qp, and reaches an asymptotic level.

474

LEGEND Spr15rnM Spr05mM .5-L 0

sm0.0 n-M 200

400

600

600

illumination

1000

1200

1400

1

0

time , s

Fig. 7. Fluorescence intensity of ‘I’rp solutions (3.5 X lop3 M) as a function during continuous and constant illumination of the samples in the fluorometer.

of time

LEGEND

-----

SprlOmt.4 Spr05nlM

o-! . 510

520

.

530

.

540

.

550

.

560

.

570

.

580

.

590

.

600

.

610

SW 0.0 mM f !O

Wavelength , nm Fig. 8. Fluorescence emission spectra of the photoproducts formed in 3.5 X 10d3 M ‘I’rp solutions subjected to equal irradiation conditions (280 nm, 300 s irradiation time). The excitation wavelength is 350 nm.

The peak-to-peak height of the half-field spectrum of the frozen solution, measured during continuous irradiation of the sample in the cavity (see Fig. 9(B)), has the same dependence on Spr addition (in the initial range of Spr additions) as the full-field spectrum (Fig. 9(A)) and vP.

475 3-

A 2. :

... . ‘, .

1.

0. :

. : -1.

.. ’ :

LEGEND .

‘$3

328

333

338

. .. . . . Gprwnu

L Finwnv 343

Magn. field , mTesla

Magn. field , mTesla Fig. 9. (A) EPR spectrum of a frozen aqueous Trp solution (3.5 x 10m3 M) after 5 min of irradiation (285 nm, 4 mW cmm2). Microwave power attenuation, 15 dB; microwave frequency, 9.43 GHz; gain of the phase-sensitive amplifier, 2.5 X 10’; modulation width, 0.15 mT. The presence of Spr increases the number of Trp’ radicals produced. (B) The half-field EPR spectrum of the same solution as in (A), using the same experimental arrangement but continuous irradiation (gain, 6 X 10’; modulation, 0.5 mT).

i-iik~5-t

&X;?

In;

O ≺3 rn;

2o

O &)r;

rn;

2o

Fig. 10. Relative yields of photoproduct (left), tryptophyl radical (middle) and triplet state (right) (measured by electron paramagnetic resonance (EPR)) in photoirradiated samples of Trp as a function of [Spr] ([Trp] = 3.5 X 10m3 M, hb, = 280 nm). The yield of the photoproduct is measured as the height of the maximum of the fluorescence spectrum and the yield of the radicals is measured as the peak-to-peak height of the EPR signal.

476

Both the rise and decay times of the phosphorescence signal, measured as the half-field EPR signal, are approximately equal. The rise time is 3.5 s and the decay time is 4.3 s (l/e fraction of the steady state value). Furthermore, the phosphorescence lifetime is not sensitive to changes in [Spr] (data not shown).

4. Discussion The linear relationship between the luminescence and the exciting fluence rate for all Trp concentrations indicates that the photophysical processes in Trp at these intensities are monophotonic. When Spr is added to solutions of low Trp concentration, q, varies in the opposite manner to qf. The relative changes in the two quantities are the same when they are scaled to unity prior to the Spr additions. Both of these findings are consistent with the hypothesis that electron ejection occurs from an excited state [4, 111 (possibly ‘L,) in the singlet manifold above S,, from which fluorescence and intersystem crossing occur. When this state has some charge-transfer (CT) character we can write its wavefunction tiCT as h2T(1La)

= a$s

+ wb+*-

where a and b are mixing coefficients and lclsis a singlet state wavefunction. D is the electron donor (Trp) and A is the acceptor (a water molecule or a solution shell) [ 111. The complex formed between Trp and Spr seems to reduce the amount of CT character (the b coefficient is less) of the nonrelaxed (‘L,) state in the singlet state manifold. The same percentage changes, in opposite directions, are observed in qf and rl,. The photodegradation is assumed to be initiated by the charge separation (the $n+A_ state) and hence it should be proportional to b2. The fluorescence, originating from the S1 state below ‘L,, should vary according to u2. Owing to the normalization of the wavefunction, the sum of a2 and b2 is unity, and the measurements are in line with an expression of this type. The actual dependence of nf on [Spr] reveals that an association complex is formed at low concentration K, Trp + 2Spr t----,

Trp-Spr,

The ratio of the concentration of the complex Trp-Spr2 to that of the Trp monomer is

lTrp-Spr21/[Trpl = KJ.Spr12 At [Spr] = 0 all the fluorescence from the solution is from Trp monomers and in the limiting case of high [Spr] all fluorescence is from the association complex [6]. The equilibrium constant is determined from Fig. 4 (upper) to be K, = 3.0 X lo4 Mp2. The two Spr entities in association with one Trp

477

molecule perturb the non-relaxed ‘L, state of Trp so that the degree of CT character is reduced by 65%. Spr also affects qp. At low Trp concentrations qp is influenced by two factors. Firstly, enhanced population of the SI level, due to the reduced CT character of the ‘L, state, will lead to both enhanced fluorescence and phosphorescence. Both If and Ip increase when Spr is initially added (Fig. .3). However, when larger amounts of Spr are added Ip declines but If does not. A second effect of the formation of the association complex, in addition to reducing the CT nature of the ‘L, state, is either to reduce the degree of intersystem crossing or to enhance the rate constant of non-radiative relaxation of the triplet state. The quenching of the Trp phosphorescence at higher concentrations can be explained by increased stretching of the NH bond of the imino group of the indole ring [ 91. An increase in this bond length will also cause a simultaneous blue shift of the fluorescence spectrum. At high concentrations the imino hydrogen on one Trp molecule (denoted Trp,) may take part in the formation of a hydrogen bond with a nitrogen (or oxygen) atom on a neighbouring Trp molecule (denoted Trp,) [9]

The two interacting Trp molecules (in the above structures the rest of the Trp molecule is denoted by R) that form this ground state complex have different spectroscopic properties. Their phosphorescence spectra ere separated by 10 nm and the long-wavelength spectrum is more slowly quenched when [Trp] is increased and is sensitive to Spr addition (Figs. 1 and 4). The different spectroscopic properties may originate from the fact that different groups of the molecules take part in the formation of the hydrogen bond which may produce different microsurroundings. The stoichiometry of the tryptophan-spermine system at high Trp concentration (3.5 X 10m3 M) can be written TW,-TrPb

+ SPr -

Kd Trpa-~b-Spr

The association complex between these two Trp molecules (called a Trp dimer, Trp,-Trpb) interacts strongly with one Spr molecule and a trimer (Trp,-Trpb-Spr) is formed (referred to as the high concentration complex) with high efficiency. The apparent equilibrium constant is Kd = 2.0 X lo3 M-l (Fig. 4, lower). The action of Spr on the Trp dimer is very different from the low concentration case where Trp exists as monomers in the solution. Spr interacts selectively with one of the Trp molecules constituting the dimer. During this

interaction the degree of hydrogen bonding of the imino group is reduced and hence the phosphorescence is restored [9]. The phosporescence is enhanced due to an increase in the rate constant of intersystem crossing. This also explains the slight reduction in qf when Spr is added. The lifetime of the triplet state, measured by the phosphorescence decay, is not affected by Spr. This observation excluded the possibility that the enhanced phosphorescence is due to changes in the rate constant of non-radiative transitions from the triplet state competing with phosphorescence. At high concentration, where Trp dimers are present in large excess compared with monomers, the Trp’ radical is mainly formed via the triplet state. The population of the triplet state, measured by phosphorescence and EPR, and the yield of Trp’ radicals have exactly the same dependence on Spr addition; hence the triplet state is a necessary precursor to the Trp’ radiCd.

At high concentration the photodegradation of Trp can be described as a sum of a fast (f) and slow (s) exponential decay [Trp] = Af exp(--kft)

+ A, exp(--h,t)

The existence of a fast exponential decay always coincides with Trp in a dimeric form. Addition of Spr enhances Trp photodegradation and radical formation by increasing intersystem crossing. The triplet state in one of the Trp units of the dimer is the starting point for hydrogen abstraction, whereby the tryptophyl radical is formed. The rate of Trp photodegradation, measured from the slopes of the double exponential decay, is 40 times higher from the triplet state than from the singlet state (‘L,). In summary the photoreactions that occur in this system can be written as WI Trp’+(D) + e-

Trp -%

T

Trp* (IL,) -

Trp*(S,) ISC

1 Trp*(T,) -

Trp’(D) + H’

At low concentrations Spr acts as an antioxidant of Trp. The degree of CT character of the excited ‘L, state in Trp is reduced by the interactions which take place in the 1:2 complex of Trp and Spr. At high Trp concentration a 2:l complex is formed between Trp and Spr. When complexed to one of the two Trp molecules, Spr acts as a pro-oxidant. Prior to any addition of Spr the phosphorescence from the Trp dimer is highly quenched. Linking of Spr to the dimer enhances the intersystem crossing (ISC) in one of the two Trp molecules of the dimer. Hence the triplet state is populated; the triplet state of Trp in this 2:l complex is a precursor for efficient radical formation. Thus Spr is a sensitizer of a Type I photoreaction process in Trp at high concentrations.

479

Even if these conclusions are restricted to the present system (Trp and Spr in aqueous salt solutions) they have a general interest. These results clearly show that the tryptophyl radical is produced both from an excited singlet and triplet state and that the population of the triplet state depends mainly on the concentration of Trp itself.

References 1 V. B. Il’yasova, Ye. P. Busel, E. A. Burshtein and A. 0. Azizova, Investigation of the photosensitized destruction of glycine in the presence of tryptophan by methods of electron paramagnetic resonance and luminescence, Biofizika, 15 (1970) 265 - 273. 2 R. Santus, M. Bazin and M. Aubailly, Nature, identification, and properties of intermediates produced by UV excitation of indole derivatives at low and room temperatures. Some applications to tryptophan-containing proteins, Rev. Chem. Zntermed., 3 (1980) 231 - 283. 3 J. F. Baugher and L. T. Grossweiner, Photolysis mechanism of aqueous tryptophan, J. Phys. Chem., 81 (1977) 1349 - 1354. 4 J. C. Mialocq, E. Amouyal, A. Bernas and D. Grand, Picosecond laser photolysis of aqueous indole and tryptophan, J. Phys. Chem., 86 (1982) 3173 - 3176. 5 T. Tamaki, Steady-state kinetic studies on the exciplex formation between indoles and n-butyl alcohol, J. Phys. Chem., 87 (1983) 2383 - 2386. 6 M. S. Walker, T. W. Bednar and R. Lumry, Exciplex studies. II. Indole and derivatives, J. Chem. Phys., 47 (1967) 1020 - 1028. 7 E. Gudgin, R. L. Delgado and W. R. Ware, Photophysics of tryptophan in HrO, DzO, and in nonaqueous solvents, J. Phys. Chem., 87 (1983) 1559 - 1565. 8 F. Bishai, E. Kuntz and L. Augenstein, Intra- and intermolecular factors affecting the excited states of aromatic amino acids, Biochim. Biophys. Acta., 140 (1967) 381 394. 9 S. V. Konev, Fluorescence and Phosphorescence of Proteins and Nucleic Acids, Plenum, New York, 1967. 10 D. Creed, The photophysics and photochemistry of near-UV absorbing amino acids - I. Tryptophan and its simple derivatives, Photochem. Photobiol., 39 (1984) 537 - 562. 11 T. B. Truong, Charge transfer to a solvent state. Luminescence studies of tryptophan in aqueous 4.5 M CaClz solutions at 300 and 77 K, J. Phys. Chem., 84 (1980) 960 964. 12 H. B. Steen, Wavelength dependence of the quantum yield of fluorescence and photoionization of indoles, J. Chem. Phys., 61 (1974) 3997 - 4002. 13 S. S. Cohen, Some roles of polyamines in microbial physiology, Adu. Enzyme Regul., 10 (1971) 207 - 223. 14 J. W. Wyse and D. A. Butterfield, Electron spin resonance and biochemical studies of the interaction of the polyamine, spermine, with the skeletal network of protines in human erythrocyte membranes, Biochim. Biophys. Acta, 941 (1988) 141 - 149. 15 B. Tadolini, Polyamine inhibition of lipoperoxidation, Biochem. J., 249 (1988) 33 - 36. 16 6. Tadolini, L. Cabrini, L. Landi, E. Varani and P. Pasquali, Polyamine binding to phospholipid vesicles and inhibition of lipid peroxidation, Biochem. Biophys. Res. Commun., 122 (1984) 550 - 555. 17 B. Tadolini, L. Cabrini, L. Landi, E. Varani and P. Pasquali, Inhibition of lipid peroxidation by spermine bound to phospholipid vesicles, Biogenic Amines, 3 (1985) 97 - 106.

480 18 0. A. Azizova, Photo-induced free radicals in frozen solutions of tryptophan, Biofizika, 9 (1964) 745 - 748. 19 M. T. Pailthorpe and C. H. Nicholls, Indole N-H bond fission during the photolysis of tryptophan,Photochem. Photobiol., 14 (1971) 135 - 145. 20 R. Santus, C. Helene and M. Ptak, Etude par resonance paramagnetique Blectronique et par spectrophotometrie d’absorption des processus primaires dans la photochimie de solutions aqueuses congelees. A 77 “K d’acides amines aromatiques et de polypeptides, Photo&em. Photobiol., 7 (1968) 341 - 360. 21 M. T. Pailthorpe, J. P. Bonjouir and C. H. Nicholls, The photolysis of tryptophan in the presence of oxygen, Photochem. Photobiol., 17 (1973) 209 - 223. 22 J. Moan and H. B. Steen, Photoinduced trapped electrons in rigid polar solution. I. A study of the recombination luminescence, J. Phys. Chem., 75 (1971) 2887 2892. 23 Y. Lion, M. Kuwabara and P. Riesz, Spin-trapping and ESR studies of the direct photolysis of aromatic amino acids, dipeptides, tripeptides and polypeptides in aqueous solutions - III. Tryptophan and related compounds, Photochem. Photobiol., 35 (1982) 53 - 62. 24 M. Hoebeke, E. Gandin and Y. Lion, Photoionization of tryptophan: an electron spin resonance investigation, Photochem. Photobiol., 44 (1986) 543 - 546. 25 J. Moan and 0. Kaalhus, Ultraviolet- and X-ray-induced radicals in frozen polar solutions of L-tryptophan, J. Chem. Phys., 61 (1974) 3556 - 3566.

The effect of the antioxidant spermine on the photophysical reactions of tryptophan in aqueous solution at 77 K.

Fluorescence, phosphorescence and electron paramagnetic resonance techniques were used to investigate the effect of the antioxidant spermine on the in...
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