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Porphyrin-melanin interaction: Effect on fluorescence and non-radiative relaxations
69
J. Photochem. Photobiol. B: Biol., 21 (1993) 6%76
Porphyrin-melanin interaction: non-radiative relaxations A. Losit,
R. Bedotti,
L. Brancaleon
effect on fluorescence
and
and C. Viappiani
Department of Physics, University of Parma, Vile delle Scienze, 43100 Parma (Italy) (Received April 14, 1993; accepted June 29, 1993)
Abstract Optical techniques and pulsed-laser, time-resolved photoacoustics (PA) were employed to obtain information on the mechanism of interaction between cationic zinc tetrabenzilpyridilporphyrin (ZnTBzPyP) and synthetic L-Dopa melanins. Synthetic eumelanin and pheomelanin strongly quench the fluorescence of ZnTBzPyP, but Stern-Volmer plots suggest a mechanism of interaction quite different for the two pigments. This diversity was confirmed by PA: for eumelanin no thermal relaxation was observed other than prompt heat, whereas for the complexed form of ZnTBzPyP with pheomelanin we were able to detect a heat-emitting species with a non-radiative lifetime in the microsecond range. The involvement of oxygen in the photophysics of the complexes formed between the cationic porphyrin and the two pigments was demonstrated, but its role has yet to be described.
Key words: Photoacoustics;
Melanin;
Porphyrin
1. Introduction During recent years, increasing interest has been devoted to the photodynamic therapy of various tumours and, in particular, malignant melanoma, a skin cancer whose incidence is becoming more widespread. Porphyrins are some of the most interesting photoactive drugs in this field and therefore a detailed knowledge of their transport and uptake in melanoma tissue, as well as their binding to macromolecules, in particular to melanoma melanins, is a necessary prerequisite in order to understand their mode of action at a subcellular level. The photochemical behaviour of porphyrins after binding to melanins deserves particular interest because, even if melanins are not the target for the therapeutic action, the strength of the porphyrin-pigment interaction suggests a potential source of photophysical side processes in addition to the main effect. A few significant contributions to these problems have been published, starting with the photochemical study of Bielec et al. [ 11. Melanin binding of some porphyrins has been investigated using solid-state 13C nuclear magnetic resonance (NMR) [2]. The formation complexes between melanins and porphyrins has +Author to whom correspondence
loll-1344/93/$6.00
should be addressed.
been studied by Ito et al. [3] using absorption and fluorescence spectroscopy. In this work, we investigate the porphyrin-melanin interaction using optical and photocalorimetric techniques. Fluorescence techniques provide valuable information on the binding equilibrium by considering the radiative de-excitation of the complex. Pulsedlaser, time-resolved photoacoustics reveals the “dark side” of the de-excitation processes, allowing the characterization of the non-radiative pathway during the relaxation of the porphyrin excited states. The picture which emerges from the experimental data is rather complex and further studies involving the use of temporal resolution in the microsecond and millisecond range would be helpful.
2. Materials
and methods
All measurements were performed in phosphate buffer (10 mM, pH 7) at room temperature. 2.1. Porphyrin The porphyrin used was zinc tetrabenzilpyridilporphyrin (ZnTBzPyP), prepared by Professor
0 1993 - Elsevier
Sequoia.
All rights reserved
70
A. Losi et al. I Pophyrin-melanin
G. Azzellini of the Institute of Chemistry, versity of Sao Paulo; Brazil.
Uni-
interaction
3. Theory
3.1. Static quenching 2.2. Melanins Eumelanin was prepared by the autooxidation of 0.5 g L-3-4-dihydroxyphenylalanine (L-Dopa) in 150 ml of water; the pH was raised to 9 and the solution was stirred for 48 h; after acidification the solution was centrifuged and the pellet dried at 40 “C; the powder thus obtained was soluble in phosphate buffer at pH 7 [4]. Pheomelanin was synthesized using the method suggested by Ito [5]. L-Dopa (150 mg) was dissolved in 150 ml of phosphate buffer at pH 7, followed by the addition of 12 mg of tyrosinase and, after 30 s, 190 mg of cysteine. The solution was stirred for about 24 h and then centrifuged after lowering the pH to a value of about 3; the pellets was resuspended in phosphate buffer at pH 7 and filtered with a 0.5 pm Millipore filter. Both fluorescence and photoacoustic measurements were performed at optical densities below 0.2 in order to minimize inner filter effects. The concentration of porphyrin was derived from the absorbance at 320 nm [6] and was maintained in the micromolar region to avoid the dimerization of the sensitizer; deoxygenation of the samples during photoacoustic measurements was achieved by bubbling nitrogen directly inside the photoacoustic cell. 2.3. Instrumentation Sample absorbance was measured using a Jasco 7850 spectrometer and fluorescence was carried out using a Perkin-Elmer LS-50 luminescence spectrometer. Photoacoustic measurements were performed using a home-built apparatus: the light source was an XeCl excimer laser (EMG 50 Lambda Physik; pulse width, 5 ns; 308 nm), pumping a dye laser (FL 3001 Lambda Physik) operating in the UV (336-350 nm) and visible (470-510 nm) regions; the photoacoustic signal was detected by a Panametrics V-103 PZT-based pressure transducer and amplified (60 decibels) by an ultrasonic pre-amplifier; an RJ-7620 (Laser Precision Corporation) energy meter was used to monitor the laser pulse energy. The signal was recorded by a LeCroy 9450A digital oscilloscope operated at 2.5 ns per channel; the data were collected and analysed with an IBM ps/2 50 computer. A time resolution of about 10 ns can be assumed for this instrument; the upper limit of detectable heat transients is approximately 5 ps.
3.1.1. Fluorescence
The theory of the static quenching of fluorescence is well established [7-91, and in this section we only summarize the models of interest to us in describing the quenching of porphyrin fluorescence by melanin. In Table 1, we report the meaning of the symbols used. The fluorescence of a chromophore is decreased by the presence of a quencher to an extent related to the quencher concentration. Melanins bind to cationic porphyrins, strongly quenching the fluorescence of the sensitizers [3]; therefore we are concerned with the theory of static quenching, which includes different models [9]. (1) The first model considers the formation of a non-fluorescent complex between a fluorescent molecule P and a non-fluorescent molecule M; in this case the fluorescence comes entirely from unbound P P+M F -= F0
-““-PM 1 1 +&JW
(1)
which is the well-known Stern-Volmer equation for static quenching; Ks represents the association constant for P and M and [M] is the concentration of the quencher. Plotting F,/F vs. [M] yields a straight line with an intercept of unity and a slope KS. When the fluorescent ligand can interact with more than one site of the quencher, the quenching process is better described by
(2)
TABLE
1. List of symbols Frequency, wavelength of excitation Average frequency of emission Fluorescence in the absence of the quencher Fluorescence in the presence of the quencher Fluorescence quantum yield Stem-Vohner constant for static quenching “Active volume” element for static quenching Absorbance (of species Z) Fraction of prompt heat (of species I) Intersystem crossing quantum yield Non-radiative relaxation lifetime Porphyrin, melanin
A. Losi et al. / Porphyrin-melanin interaction
where K’s is the association constant for site i and f;: is the fraction of chromophore bound at the ith site; in this case the Stern-Vohner plot shows deviations from linearity. (2) A second model considers the so-called “action sphere volume”: if the chromophore enters a spherical volume surrounding the quenching molecule, usually called a sphere of action, the probability of being quenched is unity, whereas it is zero outside it. In this model the quenching process is described by F - =e -
VW
(3)
FO
where Vis proportional to the volume of the action sphere [9]; the Stem-Volmer plot curves upward. For the case of several quenching sites on the same macromolecule we can write
By real-time deconvolution of the photoacoustic signal we can obtain both QNRand rNR, the lifetime of the heat-releasing species; fast events occurring on the nanosecond or shorter time scale are integrated by the transducer and only amplitude information can be obtained; in contrast, longlived species release heat too slowly to transfer energy efficiently to the piezoelectric microphone and thus give no photoacoustic signal; when the non-radiative lifetime falls within the 10 ns to 5 /..G range both @NRand rNR can be obtained. Quenching of fluorescence generally results in increased non-radiative rate parameters. The dependence of @NR on quencher concentration is readily obtained by substituting the appropriate expressions for GF and @rzIsc into eqns. (5) or (6) [12]; when static quenching of the singlet excited state implies an enhancement of the intersystem crossing probability, we propose that the quenching process is described by @NR=l-
V,,
(5)
where the term v,,/v, accounts for the Stokes shift in the fluorescence emission. In the presence of ISC, which leads the molecule to a triplet state, the fraction of prompt heat becomes
(6) where E is the molar energy of the incident photons and E, is the energy of the triplet; more generally we can write
(7) in which the last term represents the fraction of energy used in chemical reactions yielding stable product(s) or stored by transient species [lo].
v,,
@F
vex 1+&WI
where fi has the same meaning as in eqn. (2). 3.1.2. Photoacoustics Pulsed-laser, time-resolved photoacoustics is considered complementary to fluorescence, since it provides information on the non-radiative deexcitation pathways of a molecule. For simple systems with no significant degree of intersystem crossing (ISC) and showing no photochemistry, the normalized photoacoustic signal amplitude, i.e. the fraction of absorbed energy released as prompt heat [lo], is related to the fluorescence quantum yield as [ll]
71
@SC + (1 +&[MI) -
(1 +&WI)
ET
z
(8) 3.2. The problem photoacoustics
of two absorbers in
In this work we are concerned with solutions containing compounds whose absorption bands overlap at the excitation wavelength. This is a major problem in photoacoustics since it is not possible to discriminate between the heat coming from different sources. For a sample undergoing no photochemistry, the photoacoustic signal is given by 5’=KhEh @NR(1 - 10eA)
(9) where A is the sample absorbance, (1 - lOwA) is the absorbed fraction of incident light, E,, is the incident laser pulse energy and Kh is an instrumental constant empirically determined by comparison with a reference compound [13]. When no temporal shifts are evident in the time profile of the signal, the value of @NRcan conveniently be evaluated by means of the first maximum amplitude; with small signals it is useful to determine the area below the first positive oscillation of the photoacoustic wave. When two or more absorbers are present, we consider three different expressions to describe the photoacoustic signal amplitudes St,, =&E,[
@NRI(1 - lo-“‘) + &R,( 1 - lo-“‘)] (10)
72
A. Losi et al. / Porphyrin-melanin interaction
S,,,=&Eh(l-
by, say, compound 2 we must know Al, A, and @NRlTO obtain @NRzfrom eqn. (11) we rewrite
10-A”‘) @NR1$ + @Nz*~ tot tot1 (
(11) St,, =K,E,( X
QNR1(l - lo+) + (PNRZ(l_ 1o-A*) (1- 10-A’) + (1- 10-A*) [
1
(12)
Expression (10) assumes that the signals from the two species simply add to give the total photoacoustic signal; eqn. (11) takes into consideration the filter effect that each absorbing species exerts on the other and was suggested to us by J.J. Grabowski; expression (12) was proposed by Burkey et al. [14] to describe the case of multiple absorbers in photoacoustics. The last two equations both take into account the filter effect related to the presence of the competing absorption of the second species, but assume different effects of the filter on the signal. Figure 1 reports the expected photoacoustic signal amplitude for a system of two absorbers according to these three equations; it appears that they are in good accordance at low absorbances but diverge at higher ones. Preliminary results obtained in our laboratory indicate that eqn. (11) gives the best description of the phenomena; in the following we use it to calculate the fractions of prompt heat in a two-absorber system. In order to determine the fraction of prompt heat emitted 1.2
I
/
0.6
0.8
0.4
0.0
’ 0.0
I
0.2
(
4 +@NR~AA2 tot
@NR= @NR~ A tot
1 - 10 --Atot)
0.4
1.0
*1 Fig. 1. Expected photoacoustic signal for a system of two absorbers according to eqns. (10) (circles), (11) squares and (12) (triangles); here we consider two compounds, one of which (component 1) has @,.,a= 1 while the other (component 2) has GR =O.S; the absorbance of this last component is kept constant at a value of 0.1. The three expressions show the same trend at low values of A,, but clearly differ at higher absorbances.
)
(13)
from which it is easy to calculate the unknown fraction of prompt heat.
4. Results and discussion 4.1. Synthetic eumelanin 4.1.1. Absorbance
and fluorescence
The titration of a fixed quantity of ZnTBzPyP with increasing concentrations of eumelanin resulted in the expected red shift and hypochromism of the absorbance spectrum of the dye, due to the greater delocalization of the rr electrons in the central ring of the porphyrin 131.These changes in the absorption spectrum induce a pronounced hyperchromism of the dye at 475 nm and a slight hypochromism at 343 nm; to take these effects into account we preferred to measure the absorbance of the samples directly, by recording absorption spectra vs. eumelanin concentration at a tixed porphyrin concentration. A blank was run with no porphyrin, and the absorbance of the porphyrin in the presence of eumelanin was obtained as the difference of the two spectra. The addition of eumelanin to ZnTBzPyP quenched its fluorescence, centred at about 630 nm [3]; the Stern-Volmer plot for the quenching of porphyrin fluorescence is non-linear and curves upward (see Fig. 2). The best fit for F/F0 vs. [Melanin] is with eqn. (3) yielding the values for V reported in Table 2. Performing the measurements at either 20 or 40 “C led to similar results, as expected for a static quenching process. The binding of ZnTBzPyP to eumelanin seems to be on specific sites of the macromolecule, as shown by Ito et al. [3] by means of static fluorescence; this suggests that the molecular weight distribution of melanins may play a very minor role in the quenching process, which is more probably related to the local properties of the binding site. 4.1.2. Photoacoustics
From a comparison of the fluorescence emission and the fraction of prompt heat of free porphyrin, the presence of a long-lived energy-storing species is evident. The lifetime of this state is too long to be detected, although traces of an emitting species, with a lifetime at the limit of our instru-
73
A. Losi et al. / Porphyrin-melanin interaction
7
0
1
2
3
4
5
1.0
^
3 t-i 0
kcr,
0.7
radiative pathways: GNR is lower than both QNRp and GM and on deoxygenation this effect is even stronger (see Table 3). We calculated the fraction of prompt heat of porphyrin under these conditions using eqn. (11) and found values of 0.68 and 0.41 in air- and nitrogen-saturated solutions respectively excited at 345 nm and values of 0.63 and 0.36 on excitation at 475 nm. It appears that the presence of eumelanin strongly quenches the emission of prompt heat, resulting in an increased formation of long-lived species. We investigated the titration of a fixed concentration of porphyrin with increasing amounts of melanin with excitation at 475 nm; the values of
cl-
@FP(hnI%x> _ Qi,sc+&WI eVfW
1 +K,[M]
ET E
mental capabilities, are present in some of the measurements. Table 3 shows the results of deconvolution of the photoacoustic waveforms of ZnTBzPyP in the absence and presence of eumelanin; no other sources apart from prompt heat were found. The existence of a temporal modulation in heat release was detected for air-saturated ZnTBzPyP, but the value of the measured lifetime was sufficiently small to treat the signal as prompt heat. The addition of eumelanin, whose fraction of prompt heat is close to unity, modifies the non-
which should agree better with the findings from fluorescence, does not result in a significant change in the values of the parameters, probably because of the very small value of QFp. Calculating sPNRp using eqn. (12) leads to similar results, whereas using eqn. (lo), and again fitting the points with eqn. (8) leads to a value of I& of one order of magnitude lower. This disagreement was expected since the greater the absorbance of melanin, the larger the divergence of the GNRpvalues calculated according to the three different models (see Section 3); the absorbance of the solution in this case was sufficiently high to produce a filter effect on the porphyrin photoacoustic signal. The values of the photophysical parameters obtained from photoacoustics are in good agreement with those obtained from fluorescence, indicating that our model correctly accounts for the energy balance. Our photoacoustic data show that binding of porphyrin to eumelanin results in an enhanced degree of intersystem crossing of the porphyrin to a long-lived state, undetectable by our instrument; at this stage we are unable to tell whether
TABLE
2. Quenching
results of fitting experimental
A,,=345 A,,=475
nm nm
0.4
[Melanin]
(pg/iml)
Fig. 2. Direct and reciprocal Stem-Volmer plots for the quenching of ZnTBzPyP fluorescence by eumelanin; A, was 475 nm and the porphyrin concentration was 3.8 PM. The full lines are the best fit of eqn. (3) to the data; the parameters obtained are reported in Table 2, together with the results obtained with h,, = 343 nm.
of ZnTBzPyP
fluorescence
I’= 0.27 (,ug/rnl)-’ v= 0.25 (/@Ill-’
with eumelanin:
[ZnTBzPyP] = 3.8 PM [ZnTBzPyP] = 3.8 FM
data to eqn. (3) [Melanin] = O-l.9 (&ml)-’ [Melanin] =t%4.2 (&ml)-’
74
A. Losi et al. I Pophyrin-melanin
TABLE 3. Photoacoustic parameters for ZnTBzPyP and eumelanin. In the S,, column we report the fraction of prompt heat obtained from the amplitude of the first positive oscillation of the photoacoustic signal; the @r column reports the same parameter determined by deconvolution. The last column contains the non-radiative lifetime obtained from deconvolution. The concentrations were as follows: h-=475 nm, [ZnTBzPyP] =8.5 PM, [Melanin] = 3 pg ml-‘; A,=343 nm, [ZnTBzPyP] =3.8 PM, [Melanin] = 1.6 pg ml-’ @
&lU
@I
tl;a)
&)
475
@NaM @i-&air) @+.&air) %a(deox)
0.97 0.76 0.67 0.47
0.97 0.77 0.69 0.47
13 39 41 <1
;;kaa;;!
0.98 0.77 0.77 0.55
0.99 0.78 0.78 0.59
1 12
343
%a(deoxY ‘Melanin-containing
samples.
0.8
@ NR 0.7
0.6
interaction
4.2. Synthetic pheomelanin 4.2.1. Absorbance
and fluorescence
On titration with pheomelanin, the absorbance spectrum of ZnTBzPyP underwent similar changes to those observed in the case of eumelanin: a red shift of the Soret band of the porphyrin was observed, the peak being centred at 440 nm in the absence of pheomelanin and at 451 nm at 10.5 pg ml-’ pheomelanin with [ZnTBzPyP] = 4.2 PM; the same shift was observed for the other two bands in the visible region. The fluorescence quenching results can be contrasted with those presented for eumelanin; the Stern-Volmer plots curve downward, indicating the presence of heterogeneity in the process, possibly related to the presence of different sites of binding for ZnTBzPyP on the pigment. The best fit to the experimental data was obtained with eqn. (2) (see Fig. 4) and gave the values reported in Table 4; the model, reported as a full line in the figure, assumes the presence of two distinct binding sites on pheomelanin which could reflect the presence of transient radical species on the macromolecule [15]. It is worthwhile noting that fitting with eqn. (4) also reproduces the trend of the experimental data, yielding a best fit with two distinct volumes of quenching; these findings can be compared with the results from the previous model. The quenching constants obtained using eqn. (2) are of the same order of magnitude as the volumes of quenching obtained from eqn. (4); this is due to the small melanin concentrations used in this study, which makes the exponential
0.5 0
2
4
6
8
10
[ Melanin](pg/ml) Fig. 3. Fraction of prompt heat emitted by ZnTBzPyP as a function of the concentration of added eumelanin; A, was 475 nm and the porphyrin concentration was 8 PM; the full line is the best fit of eqn. (8) to the data. Parameters from the fitting are reported in Table 3.
this long-lived state is the result of the formation of a triplet state on the porphyrin or on the melanin. Further investigations are needed to determine the identity of the long-lived species, in order to elucidate the fate of the energy stored in it. Oxygen plays a major role in this system, related to its intrinsic ability to quench triplet states of photodynamic sensitizers by energy transfer processes; deoxygenation dramatically lowered the prompt non-radiative emission of the mixed solutions, whereas for the single chromophores we did not observe any significant change.
r;‘
0.6
._.I
0
6
12
18
24
30
[Pheomelanin](pg/ml) Fig. 4. Quenching of ZnTBzPyP fluorescence by pheomelanin; A,, was 343 nm and [ZnTBzPyP] was 4.2 PM, the full line is the best fit of eqn. (2) to the experimental data, and the dotted line represents the best fit with eqn. (4). The parameters from the fitting are reported in Table 4.
A. Losi et al. / Porphyrin-melanin interaction
75
TABLE 4. Parameters from the fitting of the experimental data of ZnTBzPyP fluorescence quenching by pheomelanin; the parameters are as defined in eqns. (2) and (4); [ZnTBzPyP] was 8 pM and the pheomelanin concentration ranged between 0 and 9.5 pg ml-’ A,,=345
nm
Eqn. (2) Eqn. (4)
K6 = 5.5 X 10v3 (&ml)-’ v,=7x10-3 (/.&ml)-
K2s=l.l (&Ill)-’ v*= 1.4 (/&ml)-’
A,,=475
nm
Eqn. (2) Eqn. (4)
K;=4.1~10-~ v,=5.4x10-3
K: = 0.3 (&ml)-’ v-2= 0.4 (/.Lg/ml)- ’
very similar to the linear behaviour. The observation of two distinct values of V helps us to assess the identity of the two different binding sites for ZnTBzPyP on pheomelanin; assuming an average molecular weight of 2000 Da for pheomelanin, we obtained values for the action sphere which are of the same order as the stacking of the polymer (0.15 nm) [16] and of the group COO- [9]. The origin of heterogeneity is possibly related to the two fluorescent lifetimes of ZnTBzPyP [3], which undergoes radiative transition with a double exponential decay and a very low fluorescence quantum yield. The weight of the species having a lifetime of 1.2 ns is about 99%, while the second decay (0.026 ns) only accounts for 1% of the total fluorescence emission. Ito et al. [3] have shown that, on binding to synthetic eumelanin, only the short lifetime of ZnTBzPyP is affected, while the other remains unchanged, indicating the static nature of the process. Time-resolved fluorescence measurements of ZnTBzPyP fluorescence quenching by pheomelanin will give a definitive answer to the origin of the quenching process heterogeneity; the low sensitivity of the single-photon counting apparatus used prevented us from obtaining such decisive data. From Table 4, it is evident that one of the K values is very small (approximately 10m3 (&ml) - ‘) with respect to the other (approximately 1 (r_Ls! ml)-l); this suggests that one of the two components is mildly quenched by pheomelanin and can be considered to be unaffected by binding. It is convenient to rewrite the total fluorescence in the absence of the quencher as FIJ= Fl_k.+ FOb
F0, 1+
&[Ml
AF=Foa
KdMl 1+&M
(16)
In this way we can extract both the association constant and the fraction of unquenched fluorescence. By plotting the fluorescence data according to eqn. (16) (see Fig. 5), we find values for FOa= 0.99 and FOb= 0.01 which closely resemble the fractions of total emission from the timeresolved fluorescence of ZnTBzPyP [3]. This observation supports the idea of heterogeneity originating in the different lifetimes of the fluorescent states of ZnTBzPyP. 4.2.2. Photoacoustics ZnTBzPyP in 0.01 M phosphate buffer, in the presence of pheomelanin, has a non-radiative lifetime in the microsecond range, detectable by our photoacoustic instrument. Table 5 reports the results of the deconvolution of the photoacoustic signal from ZnTBzPyP (4.2 PM) in the presence and absence of oxygen; the best fit was obtained with a two-component decay. These data indicate that, in the presence of oxygen, the lifetime of 1.2 LG’ 0.9
0.6
(14)
where F,, is the quenchable fluorescence and Fob is the fluorescence from unquenchable molecules. In the presence of the quencher M we write F=
(&ml)-’ (&ml)_’
0.3
0.0 0
+Fot,
(15)
From eqn. (15) is easy to derive an expression for the variation of the fluorescence intensity on addition of the quencher
I
1
6
12
I
18
24
30
[Pheomelanin](pg/ml) Fig. 5. Quenching of ZnTBxPyP fluorescence by pheomelanin; data are plotted according to eqn. (16); the full line is the best fit to the experimental points, obtained at F,,=O.99, Fob =O.Ol, KS =0.34 (&ml)-‘.
76
A. Losi et al. I Porphyrin-melanin interaction
TABLE 5. Non-radiative
parameters
of ZnTBzPyP-pheomelanin
References 1 J. Bielec, G. Pilas, T. Sama and T.G. Truscott, Photochemical studies of porphyrin-melanin interaction, J. Chem. Sot., Faraday Trans. 2, 282 (1986) 1469-1474. 2 G.A. Duff, J.E. Roberts and N. Foster, Analysis of spectral changes in isotopically substituted porphyrins adsorbed on melanin surfaces by solid-state “C NMR, Melanoma Res., I (1991) 201-209. 3 AS. Ito, E.C. Azzellini, S.C. Silva, 0. Serra and A.G. Szabo, Optical absorption and fluorescence spectroscopy studies of ground state melanin-cationic porphyrin complexes, Biophys. Chem., 45 (1992) 79-89. 4 P.R. Crippa, V. Horak, G. Prota, P. Svoronos and L.J. Wolfram, Chemistry of melanin, in A. Brossi (ed.), The Alkaloids, Academic Press, 1989, pp. 253-323. 5 A.S. Ito, Optimization of conditions for preparing pheomelanin, P&n. Cell. Res., 2 (1989) 53-56. 6 M.R. Chedeckel, SK. Smith, P.W. Post, A. Pokora and D.L. Wessel, Photodestruction of pheomelanin: role of oxygen, Proc. Natl. Acad. Sci. USA, 75 (1978) 5395-5399. 7 J.B. Birks, Photophysics of Aromatic Molecules, Plenum Press, 1970, 704 pp. 8 M.R. Eftink and CA. Ghiron, Fluorescence quenching studies with proteins, Anal. Biochem., 114 (1981) 199-227. 9 J.R. Lakowicz, Princ@les of Fluorescence Spectroscopy, Wiley Interscience, New York, 1983, 496 pp. 10 S.E. Braslavsb and G.E. Heibel, Time-resolved photothermal and photoacoustics methods applied to photoinduced processes in solution, Chem. Rev., 92 (1992) 1381-1410. 11 J.R. Small, J.J. Hutchings and E.W. Small, Determination of fluorescent quantum yields using pulsed-laser photoacoustic calorimetry, in E.R. Menzel (ed.), Fluorescence Detection III, SPIE - The Society of Photo-optical Instrumentation Engineers, Bellingham, WA, 1989, pp. 26-35. 12 C. Viappiani and J.R. Small, Combined photoacoustic and fluorescent quenching studies on organic dyes, in J.R. Lakowicz (ed.), Time Resolved Laser Spectroscopr in Biochemistry III, SPIE - The Society of Photo-optical Instrumentation Engineers, Bellingham, WA, 1992, pp. 285-294. 13 J.E. Rudzki, J.L. Goodman and K.S. Peters, Simultaneous determination of photoreaction dynamics and energetics using pulsed, time-resolved photoacoustic calorimetry,./. Am. Chem. Sot., 107 (1985) 7849-7854. 14 T.J. Burkey, M. Majewski and D. Griller, Heats of formation of radicals and molecules by a photoacoustic technique, J. Am. Chem Sot., 108 (1986) 2218-2221. 1.5 A.S. Ito, unpublished results, 1992. 16 M.G. Bridelli, P.R. Crippa and F. Veozzoli, X-ray diffraction studies on melanins in lyophilized melanosomes, Pigm. Cell Res., 3 (1990) 187-191.
A,, (4
Conditions
01
~1 (ns)
@Z
72
345
Air Deoxygenated
0.74 0.70
0.41 0.11
2.7 ps 193 ns
475
Air Deoxygenated
0.38 0.42
0.82 0.27
3.1 /.&s 530 ns
[ZnTBzPyP] =4.2 PM; [Pheomelanin]
=6.11 wg ml-‘.
the detected long-lived state, possibly the triplet state of ZnTBzPyP, is about 2 ,US.The deoxygenated samples show a decrease in the decay lifetime compared with the air-saturated samples, which is rather perplexing. The lifetime of the slow heat decay of the ZnTBzPyP-pheomelanin complex makes the data very dticult to analyse since, in this case, it is not possible to treat the data as was done for eumelanin, for which no temporal shift was evident. Deconvolution of the data gives no meaningful answer in terms of decay amplitudes; nevertheless, information on the lifetimes of the different heat sources can be obtained. As a consequence, no attempt was made to perform a titration of the ZnTBzPyP photoacoustic signal with pheomelanin. Refinements are needed to understand the non-radiative photophysics of ZnTBzPyP more clearly and the different role oxygen plays in the interaction with pheomelanin.
Acknowledgments
We wish to acknowledge Professor G. Azzellini for supplying the ZnTBzPyP, Professor P. R. Crippa for comments and helpful discussions on this work and M. R. Kawamura for initial results. Finally, we acknowledge J. J. Grabowski for helpful discussions on the multiple absorber problem in photoacoustics.
J. Photochem. Photobiol. B: Biol., 21 (1993) 6%76
Porphyrin-melanin interaction: non-radiative relaxations A. Losit,
R. Bedotti,
L. Brancaleon
effect on fluorescence
and
and C. Viappiani
Department of Physics, University of Parma, Vile delle Scienze, 43100 Parma (Italy) (Received April 14, 1993; accepted June 29, 1993)
Abstract Optical techniques and pulsed-laser, time-resolved photoacoustics (PA) were employed to obtain information on the mechanism of interaction between cationic zinc tetrabenzilpyridilporphyrin (ZnTBzPyP) and synthetic L-Dopa melanins. Synthetic eumelanin and pheomelanin strongly quench the fluorescence of ZnTBzPyP, but Stern-Volmer plots suggest a mechanism of interaction quite different for the two pigments. This diversity was confirmed by PA: for eumelanin no thermal relaxation was observed other than prompt heat, whereas for the complexed form of ZnTBzPyP with pheomelanin we were able to detect a heat-emitting species with a non-radiative lifetime in the microsecond range. The involvement of oxygen in the photophysics of the complexes formed between the cationic porphyrin and the two pigments was demonstrated, but its role has yet to be described.
Key words: Photoacoustics;
Melanin;
Porphyrin
1. Introduction During recent years, increasing interest has been devoted to the photodynamic therapy of various tumours and, in particular, malignant melanoma, a skin cancer whose incidence is becoming more widespread. Porphyrins are some of the most interesting photoactive drugs in this field and therefore a detailed knowledge of their transport and uptake in melanoma tissue, as well as their binding to macromolecules, in particular to melanoma melanins, is a necessary prerequisite in order to understand their mode of action at a subcellular level. The photochemical behaviour of porphyrins after binding to melanins deserves particular interest because, even if melanins are not the target for the therapeutic action, the strength of the porphyrin-pigment interaction suggests a potential source of photophysical side processes in addition to the main effect. A few significant contributions to these problems have been published, starting with the photochemical study of Bielec et al. [ 11. Melanin binding of some porphyrins has been investigated using solid-state 13C nuclear magnetic resonance (NMR) [2]. The formation complexes between melanins and porphyrins has +Author to whom correspondence
loll-1344/93/$6.00
should be addressed.
been studied by Ito et al. [3] using absorption and fluorescence spectroscopy. In this work, we investigate the porphyrin-melanin interaction using optical and photocalorimetric techniques. Fluorescence techniques provide valuable information on the binding equilibrium by considering the radiative de-excitation of the complex. Pulsedlaser, time-resolved photoacoustics reveals the “dark side” of the de-excitation processes, allowing the characterization of the non-radiative pathway during the relaxation of the porphyrin excited states. The picture which emerges from the experimental data is rather complex and further studies involving the use of temporal resolution in the microsecond and millisecond range would be helpful.
2. Materials
and methods
All measurements were performed in phosphate buffer (10 mM, pH 7) at room temperature. 2.1. Porphyrin The porphyrin used was zinc tetrabenzilpyridilporphyrin (ZnTBzPyP), prepared by Professor
0 1993 - Elsevier
Sequoia.
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70
A. Losi et al. I Pophyrin-melanin
G. Azzellini of the Institute of Chemistry, versity of Sao Paulo; Brazil.
Uni-
interaction
3. Theory
3.1. Static quenching 2.2. Melanins Eumelanin was prepared by the autooxidation of 0.5 g L-3-4-dihydroxyphenylalanine (L-Dopa) in 150 ml of water; the pH was raised to 9 and the solution was stirred for 48 h; after acidification the solution was centrifuged and the pellet dried at 40 “C; the powder thus obtained was soluble in phosphate buffer at pH 7 [4]. Pheomelanin was synthesized using the method suggested by Ito [5]. L-Dopa (150 mg) was dissolved in 150 ml of phosphate buffer at pH 7, followed by the addition of 12 mg of tyrosinase and, after 30 s, 190 mg of cysteine. The solution was stirred for about 24 h and then centrifuged after lowering the pH to a value of about 3; the pellets was resuspended in phosphate buffer at pH 7 and filtered with a 0.5 pm Millipore filter. Both fluorescence and photoacoustic measurements were performed at optical densities below 0.2 in order to minimize inner filter effects. The concentration of porphyrin was derived from the absorbance at 320 nm [6] and was maintained in the micromolar region to avoid the dimerization of the sensitizer; deoxygenation of the samples during photoacoustic measurements was achieved by bubbling nitrogen directly inside the photoacoustic cell. 2.3. Instrumentation Sample absorbance was measured using a Jasco 7850 spectrometer and fluorescence was carried out using a Perkin-Elmer LS-50 luminescence spectrometer. Photoacoustic measurements were performed using a home-built apparatus: the light source was an XeCl excimer laser (EMG 50 Lambda Physik; pulse width, 5 ns; 308 nm), pumping a dye laser (FL 3001 Lambda Physik) operating in the UV (336-350 nm) and visible (470-510 nm) regions; the photoacoustic signal was detected by a Panametrics V-103 PZT-based pressure transducer and amplified (60 decibels) by an ultrasonic pre-amplifier; an RJ-7620 (Laser Precision Corporation) energy meter was used to monitor the laser pulse energy. The signal was recorded by a LeCroy 9450A digital oscilloscope operated at 2.5 ns per channel; the data were collected and analysed with an IBM ps/2 50 computer. A time resolution of about 10 ns can be assumed for this instrument; the upper limit of detectable heat transients is approximately 5 ps.
3.1.1. Fluorescence
The theory of the static quenching of fluorescence is well established [7-91, and in this section we only summarize the models of interest to us in describing the quenching of porphyrin fluorescence by melanin. In Table 1, we report the meaning of the symbols used. The fluorescence of a chromophore is decreased by the presence of a quencher to an extent related to the quencher concentration. Melanins bind to cationic porphyrins, strongly quenching the fluorescence of the sensitizers [3]; therefore we are concerned with the theory of static quenching, which includes different models [9]. (1) The first model considers the formation of a non-fluorescent complex between a fluorescent molecule P and a non-fluorescent molecule M; in this case the fluorescence comes entirely from unbound P P+M F -= F0
-““-PM 1 1 +&JW
(1)
which is the well-known Stern-Volmer equation for static quenching; Ks represents the association constant for P and M and [M] is the concentration of the quencher. Plotting F,/F vs. [M] yields a straight line with an intercept of unity and a slope KS. When the fluorescent ligand can interact with more than one site of the quencher, the quenching process is better described by
(2)
TABLE
1. List of symbols Frequency, wavelength of excitation Average frequency of emission Fluorescence in the absence of the quencher Fluorescence in the presence of the quencher Fluorescence quantum yield Stem-Vohner constant for static quenching “Active volume” element for static quenching Absorbance (of species Z) Fraction of prompt heat (of species I) Intersystem crossing quantum yield Non-radiative relaxation lifetime Porphyrin, melanin
A. Losi et al. / Porphyrin-melanin interaction
where K’s is the association constant for site i and f;: is the fraction of chromophore bound at the ith site; in this case the Stern-Vohner plot shows deviations from linearity. (2) A second model considers the so-called “action sphere volume”: if the chromophore enters a spherical volume surrounding the quenching molecule, usually called a sphere of action, the probability of being quenched is unity, whereas it is zero outside it. In this model the quenching process is described by F - =e -
VW
(3)
FO
where Vis proportional to the volume of the action sphere [9]; the Stem-Volmer plot curves upward. For the case of several quenching sites on the same macromolecule we can write
By real-time deconvolution of the photoacoustic signal we can obtain both QNRand rNR, the lifetime of the heat-releasing species; fast events occurring on the nanosecond or shorter time scale are integrated by the transducer and only amplitude information can be obtained; in contrast, longlived species release heat too slowly to transfer energy efficiently to the piezoelectric microphone and thus give no photoacoustic signal; when the non-radiative lifetime falls within the 10 ns to 5 /..G range both @NRand rNR can be obtained. Quenching of fluorescence generally results in increased non-radiative rate parameters. The dependence of @NR on quencher concentration is readily obtained by substituting the appropriate expressions for GF and @rzIsc into eqns. (5) or (6) [12]; when static quenching of the singlet excited state implies an enhancement of the intersystem crossing probability, we propose that the quenching process is described by @NR=l-
V,,
(5)
where the term v,,/v, accounts for the Stokes shift in the fluorescence emission. In the presence of ISC, which leads the molecule to a triplet state, the fraction of prompt heat becomes
(6) where E is the molar energy of the incident photons and E, is the energy of the triplet; more generally we can write
(7) in which the last term represents the fraction of energy used in chemical reactions yielding stable product(s) or stored by transient species [lo].
v,,
@F
vex 1+&WI
where fi has the same meaning as in eqn. (2). 3.1.2. Photoacoustics Pulsed-laser, time-resolved photoacoustics is considered complementary to fluorescence, since it provides information on the non-radiative deexcitation pathways of a molecule. For simple systems with no significant degree of intersystem crossing (ISC) and showing no photochemistry, the normalized photoacoustic signal amplitude, i.e. the fraction of absorbed energy released as prompt heat [lo], is related to the fluorescence quantum yield as [ll]
71
@SC + (1 +&[MI) -
(1 +&WI)
ET
z
(8) 3.2. The problem photoacoustics
of two absorbers in
In this work we are concerned with solutions containing compounds whose absorption bands overlap at the excitation wavelength. This is a major problem in photoacoustics since it is not possible to discriminate between the heat coming from different sources. For a sample undergoing no photochemistry, the photoacoustic signal is given by 5’=KhEh @NR(1 - 10eA)
(9) where A is the sample absorbance, (1 - lOwA) is the absorbed fraction of incident light, E,, is the incident laser pulse energy and Kh is an instrumental constant empirically determined by comparison with a reference compound [13]. When no temporal shifts are evident in the time profile of the signal, the value of @NRcan conveniently be evaluated by means of the first maximum amplitude; with small signals it is useful to determine the area below the first positive oscillation of the photoacoustic wave. When two or more absorbers are present, we consider three different expressions to describe the photoacoustic signal amplitudes St,, =&E,[
@NRI(1 - lo-“‘) + &R,( 1 - lo-“‘)] (10)
72
A. Losi et al. / Porphyrin-melanin interaction
S,,,=&Eh(l-
by, say, compound 2 we must know Al, A, and @NRlTO obtain @NRzfrom eqn. (11) we rewrite
10-A”‘) @NR1$ + @Nz*~ tot tot1 (
(11) St,, =K,E,( X
QNR1(l - lo+) + (PNRZ(l_ 1o-A*) (1- 10-A’) + (1- 10-A*) [
1
(12)
Expression (10) assumes that the signals from the two species simply add to give the total photoacoustic signal; eqn. (11) takes into consideration the filter effect that each absorbing species exerts on the other and was suggested to us by J.J. Grabowski; expression (12) was proposed by Burkey et al. [14] to describe the case of multiple absorbers in photoacoustics. The last two equations both take into account the filter effect related to the presence of the competing absorption of the second species, but assume different effects of the filter on the signal. Figure 1 reports the expected photoacoustic signal amplitude for a system of two absorbers according to these three equations; it appears that they are in good accordance at low absorbances but diverge at higher ones. Preliminary results obtained in our laboratory indicate that eqn. (11) gives the best description of the phenomena; in the following we use it to calculate the fractions of prompt heat in a two-absorber system. In order to determine the fraction of prompt heat emitted 1.2
I
/
0.6
0.8
0.4
0.0
’ 0.0
I
0.2
(
4 +@NR~AA2 tot
@NR= @NR~ A tot
1 - 10 --Atot)
0.4
1.0
*1 Fig. 1. Expected photoacoustic signal for a system of two absorbers according to eqns. (10) (circles), (11) squares and (12) (triangles); here we consider two compounds, one of which (component 1) has @,.,a= 1 while the other (component 2) has GR =O.S; the absorbance of this last component is kept constant at a value of 0.1. The three expressions show the same trend at low values of A,, but clearly differ at higher absorbances.
)
(13)
from which it is easy to calculate the unknown fraction of prompt heat.
4. Results and discussion 4.1. Synthetic eumelanin 4.1.1. Absorbance
and fluorescence
The titration of a fixed quantity of ZnTBzPyP with increasing concentrations of eumelanin resulted in the expected red shift and hypochromism of the absorbance spectrum of the dye, due to the greater delocalization of the rr electrons in the central ring of the porphyrin 131.These changes in the absorption spectrum induce a pronounced hyperchromism of the dye at 475 nm and a slight hypochromism at 343 nm; to take these effects into account we preferred to measure the absorbance of the samples directly, by recording absorption spectra vs. eumelanin concentration at a tixed porphyrin concentration. A blank was run with no porphyrin, and the absorbance of the porphyrin in the presence of eumelanin was obtained as the difference of the two spectra. The addition of eumelanin to ZnTBzPyP quenched its fluorescence, centred at about 630 nm [3]; the Stern-Volmer plot for the quenching of porphyrin fluorescence is non-linear and curves upward (see Fig. 2). The best fit for F/F0 vs. [Melanin] is with eqn. (3) yielding the values for V reported in Table 2. Performing the measurements at either 20 or 40 “C led to similar results, as expected for a static quenching process. The binding of ZnTBzPyP to eumelanin seems to be on specific sites of the macromolecule, as shown by Ito et al. [3] by means of static fluorescence; this suggests that the molecular weight distribution of melanins may play a very minor role in the quenching process, which is more probably related to the local properties of the binding site. 4.1.2. Photoacoustics
From a comparison of the fluorescence emission and the fraction of prompt heat of free porphyrin, the presence of a long-lived energy-storing species is evident. The lifetime of this state is too long to be detected, although traces of an emitting species, with a lifetime at the limit of our instru-
73
A. Losi et al. / Porphyrin-melanin interaction
7
0
1
2
3
4
5
1.0
^
3 t-i 0
kcr,
0.7
radiative pathways: GNR is lower than both QNRp and GM and on deoxygenation this effect is even stronger (see Table 3). We calculated the fraction of prompt heat of porphyrin under these conditions using eqn. (11) and found values of 0.68 and 0.41 in air- and nitrogen-saturated solutions respectively excited at 345 nm and values of 0.63 and 0.36 on excitation at 475 nm. It appears that the presence of eumelanin strongly quenches the emission of prompt heat, resulting in an increased formation of long-lived species. We investigated the titration of a fixed concentration of porphyrin with increasing amounts of melanin with excitation at 475 nm; the values of
cl-
@FP(hnI%x> _ Qi,sc+&WI eVfW
1 +K,[M]
ET E
mental capabilities, are present in some of the measurements. Table 3 shows the results of deconvolution of the photoacoustic waveforms of ZnTBzPyP in the absence and presence of eumelanin; no other sources apart from prompt heat were found. The existence of a temporal modulation in heat release was detected for air-saturated ZnTBzPyP, but the value of the measured lifetime was sufficiently small to treat the signal as prompt heat. The addition of eumelanin, whose fraction of prompt heat is close to unity, modifies the non-
which should agree better with the findings from fluorescence, does not result in a significant change in the values of the parameters, probably because of the very small value of QFp. Calculating sPNRp using eqn. (12) leads to similar results, whereas using eqn. (lo), and again fitting the points with eqn. (8) leads to a value of I& of one order of magnitude lower. This disagreement was expected since the greater the absorbance of melanin, the larger the divergence of the GNRpvalues calculated according to the three different models (see Section 3); the absorbance of the solution in this case was sufficiently high to produce a filter effect on the porphyrin photoacoustic signal. The values of the photophysical parameters obtained from photoacoustics are in good agreement with those obtained from fluorescence, indicating that our model correctly accounts for the energy balance. Our photoacoustic data show that binding of porphyrin to eumelanin results in an enhanced degree of intersystem crossing of the porphyrin to a long-lived state, undetectable by our instrument; at this stage we are unable to tell whether
TABLE
2. Quenching
results of fitting experimental
A,,=345 A,,=475
nm nm
0.4
[Melanin]
(pg/iml)
Fig. 2. Direct and reciprocal Stem-Volmer plots for the quenching of ZnTBzPyP fluorescence by eumelanin; A, was 475 nm and the porphyrin concentration was 3.8 PM. The full lines are the best fit of eqn. (3) to the data; the parameters obtained are reported in Table 2, together with the results obtained with h,, = 343 nm.
of ZnTBzPyP
fluorescence
I’= 0.27 (,ug/rnl)-’ v= 0.25 (/@Ill-’
with eumelanin:
[ZnTBzPyP] = 3.8 PM [ZnTBzPyP] = 3.8 FM
data to eqn. (3) [Melanin] = O-l.9 (&ml)-’ [Melanin] =t%4.2 (&ml)-’
74
A. Losi et al. I Pophyrin-melanin
TABLE 3. Photoacoustic parameters for ZnTBzPyP and eumelanin. In the S,, column we report the fraction of prompt heat obtained from the amplitude of the first positive oscillation of the photoacoustic signal; the @r column reports the same parameter determined by deconvolution. The last column contains the non-radiative lifetime obtained from deconvolution. The concentrations were as follows: h-=475 nm, [ZnTBzPyP] =8.5 PM, [Melanin] = 3 pg ml-‘; A,=343 nm, [ZnTBzPyP] =3.8 PM, [Melanin] = 1.6 pg ml-’ @
&lU
@I
tl;a)
&)
475
@NaM @i-&air) @+.&air) %a(deox)
0.97 0.76 0.67 0.47
0.97 0.77 0.69 0.47
13 39 41 <1
;;kaa;;!
0.98 0.77 0.77 0.55
0.99 0.78 0.78 0.59
1 12
343
%a(deoxY ‘Melanin-containing
samples.
0.8
@ NR 0.7
0.6
interaction
4.2. Synthetic pheomelanin 4.2.1. Absorbance
and fluorescence
On titration with pheomelanin, the absorbance spectrum of ZnTBzPyP underwent similar changes to those observed in the case of eumelanin: a red shift of the Soret band of the porphyrin was observed, the peak being centred at 440 nm in the absence of pheomelanin and at 451 nm at 10.5 pg ml-’ pheomelanin with [ZnTBzPyP] = 4.2 PM; the same shift was observed for the other two bands in the visible region. The fluorescence quenching results can be contrasted with those presented for eumelanin; the Stern-Volmer plots curve downward, indicating the presence of heterogeneity in the process, possibly related to the presence of different sites of binding for ZnTBzPyP on the pigment. The best fit to the experimental data was obtained with eqn. (2) (see Fig. 4) and gave the values reported in Table 4; the model, reported as a full line in the figure, assumes the presence of two distinct binding sites on pheomelanin which could reflect the presence of transient radical species on the macromolecule [15]. It is worthwhile noting that fitting with eqn. (4) also reproduces the trend of the experimental data, yielding a best fit with two distinct volumes of quenching; these findings can be compared with the results from the previous model. The quenching constants obtained using eqn. (2) are of the same order of magnitude as the volumes of quenching obtained from eqn. (4); this is due to the small melanin concentrations used in this study, which makes the exponential
0.5 0
2
4
6
8
10
[ Melanin](pg/ml) Fig. 3. Fraction of prompt heat emitted by ZnTBzPyP as a function of the concentration of added eumelanin; A, was 475 nm and the porphyrin concentration was 8 PM; the full line is the best fit of eqn. (8) to the data. Parameters from the fitting are reported in Table 3.
this long-lived state is the result of the formation of a triplet state on the porphyrin or on the melanin. Further investigations are needed to determine the identity of the long-lived species, in order to elucidate the fate of the energy stored in it. Oxygen plays a major role in this system, related to its intrinsic ability to quench triplet states of photodynamic sensitizers by energy transfer processes; deoxygenation dramatically lowered the prompt non-radiative emission of the mixed solutions, whereas for the single chromophores we did not observe any significant change.
r;‘
0.6
._.I
0
6
12
18
24
30
[Pheomelanin](pg/ml) Fig. 4. Quenching of ZnTBzPyP fluorescence by pheomelanin; A,, was 343 nm and [ZnTBzPyP] was 4.2 PM, the full line is the best fit of eqn. (2) to the experimental data, and the dotted line represents the best fit with eqn. (4). The parameters from the fitting are reported in Table 4.
A. Losi et al. / Porphyrin-melanin interaction
75
TABLE 4. Parameters from the fitting of the experimental data of ZnTBzPyP fluorescence quenching by pheomelanin; the parameters are as defined in eqns. (2) and (4); [ZnTBzPyP] was 8 pM and the pheomelanin concentration ranged between 0 and 9.5 pg ml-’ A,,=345
nm
Eqn. (2) Eqn. (4)
K6 = 5.5 X 10v3 (&ml)-’ v,=7x10-3 (/.&ml)-
K2s=l.l (&Ill)-’ v*= 1.4 (/&ml)-’
A,,=475
nm
Eqn. (2) Eqn. (4)
K;=4.1~10-~ v,=5.4x10-3
K: = 0.3 (&ml)-’ v-2= 0.4 (/.Lg/ml)- ’
very similar to the linear behaviour. The observation of two distinct values of V helps us to assess the identity of the two different binding sites for ZnTBzPyP on pheomelanin; assuming an average molecular weight of 2000 Da for pheomelanin, we obtained values for the action sphere which are of the same order as the stacking of the polymer (0.15 nm) [16] and of the group COO- [9]. The origin of heterogeneity is possibly related to the two fluorescent lifetimes of ZnTBzPyP [3], which undergoes radiative transition with a double exponential decay and a very low fluorescence quantum yield. The weight of the species having a lifetime of 1.2 ns is about 99%, while the second decay (0.026 ns) only accounts for 1% of the total fluorescence emission. Ito et al. [3] have shown that, on binding to synthetic eumelanin, only the short lifetime of ZnTBzPyP is affected, while the other remains unchanged, indicating the static nature of the process. Time-resolved fluorescence measurements of ZnTBzPyP fluorescence quenching by pheomelanin will give a definitive answer to the origin of the quenching process heterogeneity; the low sensitivity of the single-photon counting apparatus used prevented us from obtaining such decisive data. From Table 4, it is evident that one of the K values is very small (approximately 10m3 (&ml) - ‘) with respect to the other (approximately 1 (r_Ls! ml)-l); this suggests that one of the two components is mildly quenched by pheomelanin and can be considered to be unaffected by binding. It is convenient to rewrite the total fluorescence in the absence of the quencher as FIJ= Fl_k.+ FOb
F0, 1+
&[Ml
AF=Foa
KdMl 1+&M
(16)
In this way we can extract both the association constant and the fraction of unquenched fluorescence. By plotting the fluorescence data according to eqn. (16) (see Fig. 5), we find values for FOa= 0.99 and FOb= 0.01 which closely resemble the fractions of total emission from the timeresolved fluorescence of ZnTBzPyP [3]. This observation supports the idea of heterogeneity originating in the different lifetimes of the fluorescent states of ZnTBzPyP. 4.2.2. Photoacoustics ZnTBzPyP in 0.01 M phosphate buffer, in the presence of pheomelanin, has a non-radiative lifetime in the microsecond range, detectable by our photoacoustic instrument. Table 5 reports the results of the deconvolution of the photoacoustic signal from ZnTBzPyP (4.2 PM) in the presence and absence of oxygen; the best fit was obtained with a two-component decay. These data indicate that, in the presence of oxygen, the lifetime of 1.2 LG’ 0.9
0.6
(14)
where F,, is the quenchable fluorescence and Fob is the fluorescence from unquenchable molecules. In the presence of the quencher M we write F=
(&ml)-’ (&ml)_’
0.3
0.0 0
+Fot,
(15)
From eqn. (15) is easy to derive an expression for the variation of the fluorescence intensity on addition of the quencher
I
1
6
12
I
18
24
30
[Pheomelanin](pg/ml) Fig. 5. Quenching of ZnTBxPyP fluorescence by pheomelanin; data are plotted according to eqn. (16); the full line is the best fit to the experimental points, obtained at F,,=O.99, Fob =O.Ol, KS =0.34 (&ml)-‘.
76
A. Losi et al. I Porphyrin-melanin interaction
TABLE 5. Non-radiative
parameters
of ZnTBzPyP-pheomelanin
References 1 J. Bielec, G. Pilas, T. Sama and T.G. Truscott, Photochemical studies of porphyrin-melanin interaction, J. Chem. Sot., Faraday Trans. 2, 282 (1986) 1469-1474. 2 G.A. Duff, J.E. Roberts and N. Foster, Analysis of spectral changes in isotopically substituted porphyrins adsorbed on melanin surfaces by solid-state “C NMR, Melanoma Res., I (1991) 201-209. 3 AS. Ito, E.C. Azzellini, S.C. Silva, 0. Serra and A.G. Szabo, Optical absorption and fluorescence spectroscopy studies of ground state melanin-cationic porphyrin complexes, Biophys. Chem., 45 (1992) 79-89. 4 P.R. Crippa, V. Horak, G. Prota, P. Svoronos and L.J. Wolfram, Chemistry of melanin, in A. Brossi (ed.), The Alkaloids, Academic Press, 1989, pp. 253-323. 5 A.S. Ito, Optimization of conditions for preparing pheomelanin, P&n. Cell. Res., 2 (1989) 53-56. 6 M.R. Chedeckel, SK. Smith, P.W. Post, A. Pokora and D.L. Wessel, Photodestruction of pheomelanin: role of oxygen, Proc. Natl. Acad. Sci. USA, 75 (1978) 5395-5399. 7 J.B. Birks, Photophysics of Aromatic Molecules, Plenum Press, 1970, 704 pp. 8 M.R. Eftink and CA. Ghiron, Fluorescence quenching studies with proteins, Anal. Biochem., 114 (1981) 199-227. 9 J.R. Lakowicz, Princ@les of Fluorescence Spectroscopy, Wiley Interscience, New York, 1983, 496 pp. 10 S.E. Braslavsb and G.E. Heibel, Time-resolved photothermal and photoacoustics methods applied to photoinduced processes in solution, Chem. Rev., 92 (1992) 1381-1410. 11 J.R. Small, J.J. Hutchings and E.W. Small, Determination of fluorescent quantum yields using pulsed-laser photoacoustic calorimetry, in E.R. Menzel (ed.), Fluorescence Detection III, SPIE - The Society of Photo-optical Instrumentation Engineers, Bellingham, WA, 1989, pp. 26-35. 12 C. Viappiani and J.R. Small, Combined photoacoustic and fluorescent quenching studies on organic dyes, in J.R. Lakowicz (ed.), Time Resolved Laser Spectroscopr in Biochemistry III, SPIE - The Society of Photo-optical Instrumentation Engineers, Bellingham, WA, 1992, pp. 285-294. 13 J.E. Rudzki, J.L. Goodman and K.S. Peters, Simultaneous determination of photoreaction dynamics and energetics using pulsed, time-resolved photoacoustic calorimetry,./. Am. Chem. Sot., 107 (1985) 7849-7854. 14 T.J. Burkey, M. Majewski and D. Griller, Heats of formation of radicals and molecules by a photoacoustic technique, J. Am. Chem Sot., 108 (1986) 2218-2221. 1.5 A.S. Ito, unpublished results, 1992. 16 M.G. Bridelli, P.R. Crippa and F. Veozzoli, X-ray diffraction studies on melanins in lyophilized melanosomes, Pigm. Cell Res., 3 (1990) 187-191.
A,, (4
Conditions
01
~1 (ns)
@Z
72
345
Air Deoxygenated
0.74 0.70
0.41 0.11
2.7 ps 193 ns
475
Air Deoxygenated
0.38 0.42
0.82 0.27
3.1 /.&s 530 ns
[ZnTBzPyP] =4.2 PM; [Pheomelanin]
=6.11 wg ml-‘.
the detected long-lived state, possibly the triplet state of ZnTBzPyP, is about 2 ,US.The deoxygenated samples show a decrease in the decay lifetime compared with the air-saturated samples, which is rather perplexing. The lifetime of the slow heat decay of the ZnTBzPyP-pheomelanin complex makes the data very dticult to analyse since, in this case, it is not possible to treat the data as was done for eumelanin, for which no temporal shift was evident. Deconvolution of the data gives no meaningful answer in terms of decay amplitudes; nevertheless, information on the lifetimes of the different heat sources can be obtained. As a consequence, no attempt was made to perform a titration of the ZnTBzPyP photoacoustic signal with pheomelanin. Refinements are needed to understand the non-radiative photophysics of ZnTBzPyP more clearly and the different role oxygen plays in the interaction with pheomelanin.
Acknowledgments
We wish to acknowledge Professor G. Azzellini for supplying the ZnTBzPyP, Professor P. R. Crippa for comments and helpful discussions on this work and M. R. Kawamura for initial results. Finally, we acknowledge J. J. Grabowski for helpful discussions on the multiple absorber problem in photoacoustics.