Paper 8 BSA
FULL PAPER
PPS
Abimbola Ogunsipe and Tebello Nyokong*
Department of Chemistry, Rhodes University, Grahamstown, 6140, South Africa
www.rsc.org/pps
Photophysicochemical consequences of bovine serum albumin
binding to non-transition metal phthalocyanine sulfonates
Received 25th October 2004, Accepted 9th May 2005
First published as an Advance Article on the web 19th May 2005
DOI: 10.1039/b416304d
The interactions between bovine serum albumin (BSA) and sulfonated metallophthalocyanine (MPc) complexes of
aluminum (AlPcSmix ), zinc (ZnPcSmix ), silicon (SiPcSmix ), germanium (GePcSmix ) and tin (SnPcSmix ) are studied using
fluorescence quenching of BSA by MPc complexes. The fluorescence quantum yields of the non-aggregated MPc
complexes (AlPcSmix , GePcSmix and SiPcSmix ) decreased in the presence of BSA, but increased for the aggregated
ZnPcSmix and SnPcSmix complexes. The BSA: MPc conjugates were less stable than the corresponding MPc complexes.
The quenching constants were much higher for the non-aggregated complexes. The aggregated nature of the
complexes also affected the rate constants (kF , kIC , kISC ) for the deactivation of the excited singlet state.
510
Introduction
Experimental
The chemistry of phthalocyanines (Pcs) has continued to gain
popularity in the last few decades because of its richness and
the extensive applications of these compounds. The robust
chemistry of Pcs is exploited in their use as photocatalysts. The
existence of a transient but relatively long-lived excited state
is vital in photocatalysis. Phthalocyanine complexes of Al(III),
Si(IV), Ge(IV), Sn(IV) and Zn(II) are favoured as good candidates
for consideration in photocatalysis and photosensitization, due
to the fact that these metal ions are diamagnetic and are expected
to give relatively long-lived excited states. The excited singlet
states of MPcs are short-lived (a few nanoseconds) and so this
state is usually not considered for photocatalysis. The excited
triplet states on the other hand have lifetimes in the microto milli-second range, which is long enough for a variety of
quenching processes to compete favourably with phosphorescence. Sulfonated aluminium phthalocyanine derivatives have
been used successfully as photosensitisers in photodynamic
therapy (PDT)1–3 and there is an aggressive on-going effort to
develop other non-transition metal phthalocyanine sulfonates
for the same purpose.
After injection into the blood stream, a PDT drug is prone to
encounter serum proteins, which are known to constitute a major
component of blood hence influences drug distribution. Bovine
serum albumin (BSA) has been used widely4–6 for binding studies
because it has been extensively characterized. The investigation
of binding of porphyrin-like drugs with albumin is of interest
and much energy has been invested into it.7–10 Serum albumins
display effective drug delivery functions,7,11 therefore a study
of their binding to porphyrin-based drugs is of significance.
Since the intrinsic fluorescence of proteins is usually quenched
upon binding to tetrapyrrolic compounds,12 this spectroscopic
behaviour provides a means of studying the interaction between
these compounds and BSA. Interactions between BSA and MPc
complexes are known13,14 and interaction between sulfonated
ZnPc complex and BSA resulted in conformational change of
BSA.15 Fluorescence from the tryptophan residues in BSA has
been reported to be quenched in the presence of phthalocyanine
derivatives containing Si, Zn or Al central metals.13,15,16 In this
work, we report the binding of sulfonated phthalocyanine complexes of Al, Si, Ge, Sn and Zn to BSA; and the photophysical
and photochemical consequences of the binding. The effect of
central metal on these properties is explored.
Materials
Photochem. Photobiol. Sci., 2005, 4, 510–516
Aluminium (AlPcSmix), silicon (SiPcSmix), germanium (GePcSmix),
tin (SnPcSmix ) and zinc (ZnPcSmix ) phthalocyanine complexes containing differently substituted sulfophthalocyanines
were synthesised from the ClAlPc, (OH)2 SiPc, (OH)2 GePc
(OH)2 SnPc and ZnPc, respectively, using fuming sulfuric acid
(30% SO3 ) according to the reported procedures for AlPcSmix .17,18
The starting materials, (OH)2 SiPc, (OH)2 GePc and (OH)2 SnPc,
were prepared, purified and characterised according to literature procedures.19 ZnPc and ClAlPc complexes were obtained
from Sigma-Aldrich. The MPcSmix complexes are known1
to contain a mixture of the di-, tri-, and tetra-sulfonated
derivatives with an average of three sulfonate groups per
molecule (Fig. 1). High pressure liquid chromatography was
employed to characterize the mixture. BSA was purchased from
FLUKA and phosphate buffer solution (PBS, pH 7.4) was used
as solvent.
Fig. 1
Molecular structure of MPcSmix . M = Al, Si, Zn, Ge or Sn.
Equipment
Absorption spectra were recorded on a Varian Cary 500 UV-VisNIR spectrophotometer. Fluorescence excitation and emission
spectra were recorded on a Varian Eclipse spectrofluoremeter.
Light intensities were measured with a POWER MAX5100
(Molelectron detector incorporated) power meter. Triplet lifetimes and yields were recorded on a flash photolysis system,
with excitation pulses generated by a Nd:YAG laser (QuantaRay, 1.5 J/8 ns)-pumped dye laser (Lambda Physic FL 3002,
This journal is
© The Royal Society of Chemistry and Owner Societies 2005
Pyridine 1 dye in methanol). The analyzing beam source was
from a Thermo Oriel xenon arc lamp, and photomultiplier tube
was used as a detector. Signals were recorded with a two-channel
digital real-time oscilloscope (Tektronix TDS 360). High pressure liquid chromatography (HPLC) was performed on a QuadGradient HPLC system, Agilent 1100 Series; fitted with an
analytical column, l Bondapak C18 (390 × 3.00 mm) and
connected to a variable wavelength UV-Vis detector (set at k =
365 nm). The mobile phase comprised of 50:50 methanol:water
mixture, with a flow rate of 1 ml min−1 and sample injection
volume of 20 ll.
Photodegradation methods
Photodegradation (U d ) quantum yield determinations were carried out using the experimental set-up and equations described
in detail elsewhere.20–22 Typically, a 2 ml solution of MPcSmix
(absorbance ∼1.0 at the irradiation wavelength, the Q band) was
irradiated using a General electric Quartz line lamp (300 W).
A 600 nm glass cut off filter (Schott) and a water filter were
used to filter off ultraviolet and infrared radiations respectively.
An interference filter (Intor, 670 nm with a band width of
20 nm) was additionally placed in the light path before the
sample. Light intensity used for the experiments was 4.82 ×
1016 photons s−1 cm−2 .
MPcSmix binding to bovine serum albumin
The binding of the MPcSmix complexes to BSA was studied by
spectrofluorometry at room temperature (25 ◦ C). A solution of
BSA in PBS 7.4 was titrated with increasing concentrations of
the respective MPcSmix solution. BSA was excited at 280 nm
and fluorescence recorded between 290 and 500 nm. The
steady diminution in BSA fluorescence intensity with increase in
MPcSmix concentration was noted and used in the determination
of the binding constants and the number of binding sites on
BSA, according to eqn. (4).28–30
(F0 − F)
= log Kb + n log [MPcS]
(4)
log
(F − F∞ )
where F 0 and F are the fluorescence intensities of BSA in
the absence and presence of MPcSmix respectively; F ∞ , the
fluorescence intensity of BSA saturated with MPcSmix ; K b , the
binding constant; n, the number of binding sites on a BSA
molecule;
and
[MPcSmix ], the concentration of MPcSmix . Plots
(F0 −F)
of log (F−F
against log[MPcSmix ] would provide the values of
∞)
n (from slope) and K b (from the intercept).
For photophysical and photochemical studies, a molar ratio
of 10 : 1 (BSA : MPcSmix , with [BSA] = 7.5 × 10−5 M) was used.
This ratio was used due to the abundance of proteins within
most biological systems.
Fluorescence quantum yields
Fluorescence quantum yields (U F ) were determined by the
comparative method23,24 [eqn. (1)],
FAStd g2
U F = U F (Std)
FStd Ag2Std
(1)
where F and F Std are the areas under the fluorescence curves
of the MPcSmix and the reference, respectively. A and AStd are
the absorbances of the sample and reference at the excitation
wavelength respectively, and g and gStd are the refractive indices
of solvents used for the sample (in water, g = 1.330) and
reference (in ether, g = 1.352), respectively. Chlorophyll a in ether
(U F = 0.32)25 was employed as a reference. Both the sample and
reference were excited at the same wavelength. The absorbance
of the solutions at the excitation wavelength ranged between
0.04 and 0.05.
Triplet lifetimes and quantum yields
The deaerated solutions of the respective MPcSmix complexes
were introduced into a 2 mm × 10 mm spectrophotometric
cell and irradiated at the Q band with the laser system
described above. Triplet quantum yields (U T ) of the MPcSmix
complexes were determined by the singlet depletion method.20
A comparative method20,26 using zinc phthalocyanines tetrasulfonate (ZnPcS4 ) as standard was employed for the calculations,
eqn. (2).
U
Sample
T
=U
Std
T
DASample
eStd
S
S
Std Sample
DAS eS
MPcSmix fluorescence lifetimes were determined by monitoring
the fluorescence intensity as a function of quencher concentration. Here, two sets of data were obtained from: (i)
quenching of BSA fluorescence by MPcSmix and (ii) quenching of
MPcSmix fluorescence by BSA. For (i), the quenching of BSA by
MPcSmix , the changes in BSA fluorescence intensity were related
to MPcSmix concentrations by the Stern–Volmer relationship
[eqn. (5)]:
F0BSA
= 1 + kBSA
SV [MPcSmix ]
F BSA
(5)
and kBSA
SV is given by eqn. (6):
BSA
kBSA
SV = kQ sF
(6)
and F BSA are the fluorescence intensities of BSA in the
where F BSA
0
absence and presence of MPcSmix respectively; kBSA
SV , the Stern–
Volmer quenching constant; kQ , the bimolecular quenching
BSA
is
constant; and sBSA
F , the fluorescence lifetime of BSA. sF
obtained
estimated to be 10−8 s,10 thus from the values kBSA
SV
F BSA
from the plots of F0BSA versus [MPcSmix ], the value of kQ may be
determined from eqn. (6). For quenching of MPcSmix by BSA,
similar eqns. (7) and (8) apply.
F0MPc
= 1 + kMPc
SV [BSA]
F MPc
(7)
(2)
and DAStd
are the changes in the singlet state
where DAsample
S
S
absorbance of the MPcSmix complex and ZnPcS4 , respectively.
and eStd
are the singlet state extinction coefficients for the
esample
S
S
is the triplet
MPcSmix complex and ZnPcS4 , respectively. U Std
T
quantum yield for the standard ZnPcS4 in aqueous solution
(U T = 0.56).27
Quantum yields of internal conversion (U IC ) were obtained
from eqn. (3), which assumes that only three processes (fluorescence, intersystem crossing and internal conversion), jointly
deactivate the excited singlet state of an MPcSmix molecule.
U IC = 1 − (U F + U T )
Determination of MPcSmix fluorescence lifetimes
(3)
MPc
kMPc
SV = kQ sF
MPc
0
(8)
MPc
where F
and F
are the fluorescence intensities of MPcSmix
in the absence and presence of BSA respectively; kMPc
SV , the Stern–
Volmer quenching constant; kQ , the bimolecular quenching
constant; and sMPc
F , the fluorescence lifetime of MPcSmix . In both
cases, kQ is assumed to be the same. Using kMPc
SV from the plot
generated by eqn. (7), and kQ from eqn. (6), we can obtain the
from eqn. (8).
value of sMPc
F
The determination of fluorescence lifetimes afforded the
calculation of rate constants for processes deactivating the
excited singlet state, since the quantum yields are known. Values
for the rate constants for fluorescence (kF ), internal conversion
Photochem. Photobiol. Sci., 2005, 4, 510–516
511
(kIC ) and intersystem crossing (kISC ) were obtained using eqns.
(9a)–(9c):
kF =
UF
sF
(9a)
kIC =
U IC
sF
(9b)
kISC =
UT
sF
(9c)
Results and discussion
Effects of BSA binding on photophysical and photochemical
properties of MPcSmix complexes
Fig. 2 shows the ground state electronic absorption spectra of
MPcSmix mixtures in PBS 7.4. The Soret bands (not shown in
Fig. 2) were observed between 339 and 349 nm for the MPcSmix
complexes. The spectra of these complexes was the same in
unbuffered water, that is in the absence of ions present in buffered
solutions. Thus the ions in the buffered solution did not affect
the aggregation behaviour of these complexes. AlPcSmix , SiPcSmix
and GePcSmix are monomeric in nature, while ZnPcSmix and
SnPcSmix are aggregated. These observations were recently31,32
confirmed by the addition of a surfactant, Triton X-100 to these
complexes. AlPcSmix , SiPcSmix and GePcSmix gave no change in
spectra, confirming lack of aggregation whereas ZnPcSmix and
SnPcSmix gave marked enhancements of Q band intensity and
a corresponding collapse of the peak around 630 nm, which is
believed to be due to dimeric species. Aggregation in sulfonated
MPc complexes is characterised by the presence of two main
bands (instead of one for monomeric species) in the visible
region. The lower energy band is due to the monomer and the
higher energy band due to the aggregated species. The spectra
for SnPcSmix and ZnPcSmix show more monomerization at the
lower concentrations used in Fig. 2b. The presence of multiple
peaks in the Q band region of the un-aggregated SiPcSmix (shown
in Fig. 2) and GePcSmix complexes, could be an indication
of the presence of differently substituted components in the
mixture. The degree of aggregation increases with lipophilicity,
hence the prevalence of the less sulfonated fractions in solution
is expected to increase aggregation. The prevalence of less
sulfonated fractions in the aggregated ZnPcSmix and SnPcSmix
complexes and more sulfonated fractions in GePcSmix , AlPcSmix
and SiPcSmix was confirmed by HPLC, Figs. 3 and 4. From
HPLC, it is expected that the most highly sulfonated (most
soluble) would be the first to be eluted from the chromatographic
column, and so give the lowest retention time and that the less
sulfonated fractions as expected, give the highest retention times.
Thus the HPLC signals with the lowest retention times (∼1 min)
are assigned to the tetrasulfonated fractions using tetrasulfozinc
phthalocyanine (ZnTSPc) as reference. ZnPcSmix and SnPcSmix
gave similar traces (Fig. 3), with appreciable signals in the
relatively high retention time regions of the HPLC traces,
whereas GePcSmix AlPcSmix and SiPcSmix showed signals at low
retention times. These observations confirm the prevalence of the
highly sulfonated fractions in AlPcSmix , SiPcSmix and GePcSmix ,
hence less aggregation, while in ZnPcSmix and SnPcSmix , the less
sulfonated fractions are prevalent, and are more aggregated.
Fig. 3 HPLC trace for GePcSmix .
Fig. 4
Fig. 2 Ground state electronic absorption spectra of MPcSmix mixtures
in (a) PBS 7.4 (concentrations: ∼5 × 10−6 M for AlPcSmix and SiPcSmix ;
1.2 × 10−5 M for ZnPcSmix and SnPcSmix ) and (b) unbuffered water
(concentrations: ∼4 × 10−7 M for AlPcSmix and SiPcSmix; 8 × 10−7 M
for ZnPcSmix and SnPcSmix ).
512
Photochem. Photobiol. Sci., 2005, 4, 510–516
HPLC trace for ZnPcSmix .
Addition of BSA to solutions of the MPcSmix species (Fig. 5)
gave the same results as Triton X-100 addition, further confirming the aggregated nature of ZnPcSmix and SnPcSmix . The
monomerization effect of serum albumin on photosensitizers is
well documented.33–35 For the un-aggregated AlPcSmix complex,
Fig. 5 UV/Vis absorption spectral change observed on addition of
BSA to ZnPcSmix in PBS 7.4 (concentrations: ZnPcSmix = 1.63 × 10−6 M
and BSA = 1.63 × 10−5 M).
Fig. 7 Ground state electronic absorption (i) and fluorescence emission
(ii) spectra of BSA in PBS 7.4 (concentration = 2 × 10−5 M).
a small bathochromic shift (∼2 nm) in the Q band position
was observed on addition BSA, Fig. 6. This implies that a
complex is actually being formed between the photosensitizer
and the biopolymer. The lack of broadening or splitting in the
Q band, suggests that upon conjugation with BSA, AlPcSmix is
still not aggregated. A similar subtle change in Q band position
was observed for SiPcSmix , but not for GePcSmix , ZnPcSmix and
SnPcSmix . However some BSA–MPc conjugates are known not
to show shifts in the Q band compared to MPc complex alone.13
The electronic absorption and fluorescence spectra of BSA in
PBS 7.4 are shown in Fig. 7. BSA shows a strong absorption
around 280 nm (log e = 4.65) which is due to tryptophan and
tyrosine residues.4,36
Fig. 8 Fluorescence emission spectra of MPcSmix in the presence and
absence of BSA in PBS 7.4 (concentrations: AlPcSmix and ZnPcSmix =
3 × 10−6 M; BSA = 7.5 × 10−5 M).
Fig. 6 UV/Vis spectral changes observed on addition of BSA to
AlPcSmix in PBS 7.4. (concentrations: AlPcSmix = 7.5 × 10−6 M and
BSA = 7.5 × 10−5 M).
Fig. 8 shows the fluorescence emission spectra of AlPcSmix
and ZnPcSmix (free and BSA-bound). For the un-aggregated
species (AlPcSmix , SiPcSmix and GePcSmix ), BSA binding resulted
in reasonable fluorescence quenching which is manifested in decrease in the fluorescence intensity (shown in Fig. 8 for AlPcSmix )
and in fluorescence quantum yield (U F ) values (Table 1).
On the other hand, BSA binding resulted in increase in
fluorescence intensity (Fig. 8) and in U F values for the aggregated
ZnPcSmix and SnPcSmix , which further substantiates the fact that
BSA monomerizes the aggregated species, hence the increase in
fluorescence intensity rather than quenching. All the MPcSmix
complexes quenched BSA as evident from the decrease in BSA
fluorescence on titration with MPcSmix .
MPc photodegradation is believed to be a singlet oxygenmediated process,37 thus its efficiency should be related to the rate
of singlet oxygen generation, among other factors. Aggregation
is also known31 to reduce photostability, and Table 1 shows
that aggregated complexes, ZnPcSmix and SnPcSmix , show larger
degradation quantum yields. With the exception of GePcSmix for
which the value did not change (Table 1), MPcSmix photobleaching quantum yield (U d ) increased in the presence of BSA. We
attribute this increase to the formation of active oxidative
albumin species which could additionally photodegrade the
MPcSmix complex.
Table 1 Photophysical and photochemical parameters of MPcSmix complexes in PBS 7.4. Values in brackets are in the presence of 10 molar
proportions of BSA. [BSA] = 7.5 × 10−5 M
AlPcSmix
SiPcSmix
GePcSmix
ZnPcSmix
SnPcSmix
kQ /nm
kF /nm
UF
sT /ls
UT
U IC
105 /U d
UD
674
(677)
678
(679)
680
(680)
673
(676)
688
(685)
677
(683)
682
(682)
686
(686)
677
(681)
699
(688)
0.44V
(0.34)
0.34
(0.86)
0.30
(0.24)
0.16
(0.20)
0.05
(0.07)
2.93
0.44
0.12
0.42
2.90
0.45
0.21
2.76
0.68
0.03
2.95
0.53
0.31
2.52
0.59
0.36
0.40
(0.59)
0.71
(0.30)
0.45
(0.44)
3.65
(17.1)
1.59
(1.77)
U OP
U SnPcS
OP
(0.59)
0.49
(0.40)
0.68
(0.57)
0.45
(0.37)
0.42
(1.00)
Photochem. Photobiol. Sci., 2005, 4, 510–516
513
Photosensitized oxidation of BSA
MPcSmix photodegradation within the BSA–MPcSmix conjugate,
was accompanied by substantial increase in intensity in the
UV region of the spectrum (Fig. 9), implying that BSA
photodegrades in the presence of singlet oxygen, generating
products which absorb at about the same region as the original
BSA absorbing components, hence the increase in the intensity
in the UV region. Thus, BSA competes with the MPcSmix for
singlet oxygen. It is difficult to identify exactly which species is
responsible for the increase in intensity in the UV region. Due to
the relatively large concentration of BSA used in the experiments
and the high rate constants for reaction of singlet oxygen with
some amino acid side chains,38 it is logical to think that BSA
would be a target for singlet oxygen. A series of endoperoxides
and hydroperoxides have been indicated as products of amino
acid side chain oxidations in proteins.38 In order to quantify
the efficiency of BSA photooxidation, the increase in intensity
in the UV region (at 305 or 330 nm) was used, and related to
the photooxidation quantum yield (F OP ), by the usual eqn. (10)
employed for photodegradation studies:
U OP =
(At − A0 )V/e
Iabs t
(10)
where At and A0 are the absorbances at 305 or 300 nm (both
wavelength gave the same results) after irradiation for t s and
before irradiation respectively; V , the reaction volume; e, the
molar extinction coefficient of oxidation product; and I abs , the
intensity of absorbed light in photon mol s−1 .
Fig. 9 Spectral changes observed on photolysis of AlPcSmix in the
presence of BSA. pH = 7.4. Irradiation wavelength at AlPcSmix
complexes Q band maximum (674 nm). Light intensity used was 4.82 ×
1016 photons s−1 cm−1 (concentrations: AlPcSmix = 6 × 10−6 M and
BSA = 6 × 10−5 M). Irradiation time interval = 1 min.
Fig. 10 Determination of MPcSmix –BSA binding constant in PBS 7.4
(BSA concentration = 2 × 10−5 M).
Since the identity of the new species absorbing in the UV
region is not known (hence the extinction coefficient is not
known), eqn. (10) may not be used directly. We employed a
514
Photochem. Photobiol. Sci., 2005, 4, 510–516
relative method described below. The rate of formation (R) of
the oxidation products is given by eqn. (11):
R=
(At − A0 )V/e
t
(11)
Combining eqns. (10) and (11) gives eqn. (12a):
U OP =
R
Iabs
(12a)
For another photosensitizer, we can write a similar eqn. (12b):
U ′OP =
R′
′
Iabs
(12b)
Taking the ratio of eqn. (12b) over (12a), gives eqn. (13):
R′ Iabs
U ′OP
=
′
U OP
RIabs
(13)
Values of BSA photooxidation quantum yields were obtained
relative to that in SnPcSmix , which is given an arbitrary value
of 1.00. The relative values of FOP for all MPcSmix complexes
studied are listed in Table 1. Higher values were observed for Al,
Ge, and Sn complexes.
Interaction of MPcSmix with BSA
The binding constants (K b ) obtained for MPcSmix binding to
BSA, together with the binding stoichiometry of the complex
formed were obtained using eqn. (4) (Fig. 10, for SiPcSmix ); and
the results are presented in Table 2 for [BSA] = 2.0 × 10−5 M and
[MPc] ranging from 1.58 × 10−6 to 1.58 × 10−5 M. In addition
for the aggregated SnPcSmix and ZnPcSmix lower concentrations
([BSA] = 3.3 × 10−7 M and [MPc] = 1.67 × 10−7 to 8.0 × 10−7 M)
were employed. The binding contant values were the same when
unbuffered water was employed. Again showing that the salts in
the buffered solutions do not affect the aggregation behaviour of
these complexes. The values for the unaggregated Ge, Si and Al
complexes are typical of MPc–albumin interactions in aqueous
solutions,28 and did not change on dilution to low concentrations
(∼10−7 M) similar to those employed for SnPcSmix and ZnPcSmix
complexes (for binding and quenching constants shown in
brackets, Table 2). The highest value of K b was obtained for
AlPcSmix which is particularly monomeric, followed by GePcSmix
and SiPcSmix . The aggregated SnPcSmix and ZnPcSmix complexes
gave the lowest K b values, but these values increased considerably
(∼three-fold) at low concentrations as shown in Table 2. This
shows that aggregation plays an important role in the binding.
The involvement of a MPc dimer in BSA binding is indirect,
via dissociation into monomers. Consequently, K b values for
dimeric species are expected to depend largely on, and be limited
by the inherent dimer dissociation constant. The variation of
K b with the nature of the complex in Table 2 could also be
a reflection of the relative affinity of BSA for the respective
MPcSmix species. Since it is known that serum albumins have
high affinities for negatively charged molecules,39 the observed
larger values of K b for AlPcSmix , SiPcSmix and GePcSmix is a result
of the predominance of highly sulfonated derivatives as shown
by HPLC (Fig. 3). The number of binding sites on BSA (n)
obtained from the experiments is ∼1 (Table 2), which suggests a
1 : 1 stoichiometry for all the MPcSmix –BSA conjugates.
Fluorescence quenching analysis
As discussed above, BSA and each of the MPcSmix species
display mutual quenching on one another. These reciprocative
fluorescence quenching could have arisen from either dynamic
(no complexation) or static (due to non-fluorescent ground state
Table 2 Quenching and binding data for MPcSmix complexes in PBS 7.4. Unless otherwise stated: [BSA] = 2.0 × 10−5 M and [MPc] ranges from
1.58 × 10−6 to 1.58 × 10−5 M. Values in brackets are for [BSA] = 3.3 × 10−7 M and [MPc] ranging from 1.67 × 10−7 to 8.0 × 10−7 M
AlPcSmix
SiPcSmix
GePcSmix
ZnPcSmix
SnPcSmix
a
K b /10−6 M−1
n
−4
kBSA
M−1
SV /10
−3
kMPc
M−1
SV /10
kQ a /10−12 M−1 s−1
sF /ns
kF /10−7 s−1
kISC /10−7 s−1
kIC /10−7 s−1
17.21
1.29
0.81
0.10 (0.34)
0.08 (0.21)
1.4
1.3
1.1
1.0
1.0
11.45
6.90
4.54
7.36 (6.72)
1.98 (3.19)
58.43
34.36
18.48
11.40
6.90
4.54
7.36 (6.72)
1.98 (3.19)
5.13
4.98
4.07
2.927
8.58
6.83
7.37
5.52
8.58
9.04
16.71
18.28
2.34
4.22
0.74
10.69
Values determined from BSA quenching by MPcSmix .
complex) quenching. The Stern–Volmer analysis of fluorescence
data was used to discern the actual quenching mechanism.
Figs. 11 and 12 show the quenching of BSA and SiPcSmix in PBS
7.4, respectively by each other. The slopes of the plots shown
and kMPc
as inserts gave kBSA
SV
SV , respectively. Fig. 12 shows the
quenching behaviour of MPcSmix typical of the un-aggregated
complexes (Al, Si and Ge complexes). As discussed above for
the aggregated Zn and Sn complexes, there was an increase in
fluorescence intensity (rather than quenching) upon addition of
BSA, hence the kMPc
SV values could not be determined, whereas all
MPcSmix complexes could quench BSA with kBSA
SV values shown
in Table 2.
Fig. 11 Fluorescence emission spectral changes for BSA on addition
of increasing concentrations of SiPcSmix . Inset: Stern–Volmer plot for
SiPcSmix quenching of BSA. (Concentrations: BSA = 2 × 10−5 M;
SiPcSmix varies from 1.67 × 10−6 M to 1.5 × 10−5 M.)
Fig. 12 Fluorescence emission spectral changes of GePcSmix on addition of increasing concentrations of BSA. Inset: Stern–Volmer plot for
BSA quenching of GePcSmix . (Concentrations: GePcSmix = 4 × 10−6 M;
BSA varies from 1.4 × 10−5 M to 5.7 × 10−5 M.)
Values of kQ were determined from eqn. (6) for quenching
of BSA by MPc and are listed in Table 2. These values
are of the order of 1012 M−1 s−1 , Table 2. The acceptable
value of kQ for dynamic quenching according to Einstein–
Smoluchowski approximation40 at room temperature is of the
order of 1010 M−1 s−1 . The high values of kQ observed in Table 2,
suggests that the fluorescence quenching of BSA by MPcSmix is
not initiated by dynamic quenching, but by static quenching,
and kMPc
values in eqns. (6) and (8) are due to
and the kBSA
SV
SV
static quenching. These static quenching constants (kBSA
and
SV
kMPc
SV ; Table 2) for BSA and the MPcSmix complexes reveal the
relative degrees of interaction between BSA and the MPcSmix
complexes. Both constants are highest for AlPcSmix . From these
values, it could be inferred that AlPcSmix possesses the highest
degree of interaction with BSA while SnPcSmix has the lowest.
for ZnPcSmix and SnPcSmix
The observed lower values of kBSA
SV
are not unconnected with their aggregation, and the relatively
higher values for the former could imply the lower aggregation
tendency of ZnPcSmix compared to SnPcSmix . The high kBSA
SV
and kMPc
SV values for AlPcSmix compared to the other monomeric
complexes (SiPcSmix and GePcSmix ), suggests that the latter two
have lower affinities for BSA than the former.
Fluorescence lifetimes of the MPcSmix complexes (sMPc
F , Table 2) were calculated from kMPc
SV and kQ values using eqn. (8),
and the values are quite close to the typical MPc fluorescence
lifetimes.19,28 The procedure described here for calculation of sMPc
F
could however not be employed in the case of the aggregated
ZnPcSmix and SnPcSmix , whose fluorescence intensities increased
on BSA addition, hence the kBSA
SV values could not be calculated.
Among the three: AlPcSmix , SiPcSmix and GePcSmix which are
monomeric, fluorescence lifetime decreases as the atomic mass
of central metal ion increases, most probably due to an enhanced
intersystem crossing. The literature value for ZnPcSmix 27 is
notably less than those calculated for AlPcSmix , SiPcSmix and
GePcSmix (Table 2) which is attributable to the aggregated nature
of the complex.
Kinetic data
The rate constants for the excited singlet state deactivation
processes (kF , kIC and kISC ) were calculated [using eqn. (9a)–(9c)]
and are listed in Table 2. The values could not be obtained for
SnPcSmix whose sF value was not available; but among the three
unaggregated species, AlPcSmix showed the highest values of kF ,
while GePcSmix showed the lowest. Comparison of ZnPcSmix
with the rest is not appropriate due to its aggregated nature.
The values of kISC , as expected, increased with the mass of the
central metal ion, which is a manifestation of the heavy atom
effect that promotes intersystem crossing. As expected, ZnPcSmix
gave the highest value of kIC , which is again readily attributed
to aggregation. GePcSmix gave the lowest value of kIC , indicating
that this species is perhaps the most photoactive in the list. The
superior photoactivity of this species is also shown in Table 1,
where its values of U F , U T and U D are consistently high.
Conclusions
In conclusion, this work provides a qualitative and quantitative
interpretation of the interaction of MPcSmix mixtures with
BSA. Results of binding experiments showed that each of the
MPcSmix mixtures formed a 1 : 1 adduct with BSA, but the
binding feasibilities varied markedly, as evident from the values
of binding constants (K b ) for the MPcSmix –BSA adduct; with
AlPcSmix giving the highest value and SnPcSmix giving the lowest.
The observed trend in the values of K b was explained in terms of
aggregation and the greater affinities of some of the species for
BSA than others. The spectral as well as photophysicochemical
Photochem. Photobiol. Sci., 2005, 4, 510–516
515
properties of the mixtures were altered in the presence of
BSA. This work also provides an approximate but simple route
to the determination of fluorescence lifetimes of MPc complexes,
using steady-state measurements.
19
Acknowledgements
This work has been supported by the National Research
Foundation of South Africa as well as Rhodes University.
AO thanks ICSC world laboratory and Mellon foundation for
scholarships.
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PPS
Abimbola Ogunsipe and Tebello Nyokong*
Department of Chemistry, Rhodes University, Grahamstown, 6140, South Africa
www.rsc.org/pps
Photophysicochemical consequences of bovine serum albumin
binding to non-transition metal phthalocyanine sulfonates
Received 25th October 2004, Accepted 9th May 2005
First published as an Advance Article on the web 19th May 2005
DOI: 10.1039/b416304d
The interactions between bovine serum albumin (BSA) and sulfonated metallophthalocyanine (MPc) complexes of
aluminum (AlPcSmix ), zinc (ZnPcSmix ), silicon (SiPcSmix ), germanium (GePcSmix ) and tin (SnPcSmix ) are studied using
fluorescence quenching of BSA by MPc complexes. The fluorescence quantum yields of the non-aggregated MPc
complexes (AlPcSmix , GePcSmix and SiPcSmix ) decreased in the presence of BSA, but increased for the aggregated
ZnPcSmix and SnPcSmix complexes. The BSA: MPc conjugates were less stable than the corresponding MPc complexes.
The quenching constants were much higher for the non-aggregated complexes. The aggregated nature of the
complexes also affected the rate constants (kF , kIC , kISC ) for the deactivation of the excited singlet state.
510
Introduction
Experimental
The chemistry of phthalocyanines (Pcs) has continued to gain
popularity in the last few decades because of its richness and
the extensive applications of these compounds. The robust
chemistry of Pcs is exploited in their use as photocatalysts. The
existence of a transient but relatively long-lived excited state
is vital in photocatalysis. Phthalocyanine complexes of Al(III),
Si(IV), Ge(IV), Sn(IV) and Zn(II) are favoured as good candidates
for consideration in photocatalysis and photosensitization, due
to the fact that these metal ions are diamagnetic and are expected
to give relatively long-lived excited states. The excited singlet
states of MPcs are short-lived (a few nanoseconds) and so this
state is usually not considered for photocatalysis. The excited
triplet states on the other hand have lifetimes in the microto milli-second range, which is long enough for a variety of
quenching processes to compete favourably with phosphorescence. Sulfonated aluminium phthalocyanine derivatives have
been used successfully as photosensitisers in photodynamic
therapy (PDT)1–3 and there is an aggressive on-going effort to
develop other non-transition metal phthalocyanine sulfonates
for the same purpose.
After injection into the blood stream, a PDT drug is prone to
encounter serum proteins, which are known to constitute a major
component of blood hence influences drug distribution. Bovine
serum albumin (BSA) has been used widely4–6 for binding studies
because it has been extensively characterized. The investigation
of binding of porphyrin-like drugs with albumin is of interest
and much energy has been invested into it.7–10 Serum albumins
display effective drug delivery functions,7,11 therefore a study
of their binding to porphyrin-based drugs is of significance.
Since the intrinsic fluorescence of proteins is usually quenched
upon binding to tetrapyrrolic compounds,12 this spectroscopic
behaviour provides a means of studying the interaction between
these compounds and BSA. Interactions between BSA and MPc
complexes are known13,14 and interaction between sulfonated
ZnPc complex and BSA resulted in conformational change of
BSA.15 Fluorescence from the tryptophan residues in BSA has
been reported to be quenched in the presence of phthalocyanine
derivatives containing Si, Zn or Al central metals.13,15,16 In this
work, we report the binding of sulfonated phthalocyanine complexes of Al, Si, Ge, Sn and Zn to BSA; and the photophysical
and photochemical consequences of the binding. The effect of
central metal on these properties is explored.
Materials
Photochem. Photobiol. Sci., 2005, 4, 510–516
Aluminium (AlPcSmix), silicon (SiPcSmix), germanium (GePcSmix),
tin (SnPcSmix ) and zinc (ZnPcSmix ) phthalocyanine complexes containing differently substituted sulfophthalocyanines
were synthesised from the ClAlPc, (OH)2 SiPc, (OH)2 GePc
(OH)2 SnPc and ZnPc, respectively, using fuming sulfuric acid
(30% SO3 ) according to the reported procedures for AlPcSmix .17,18
The starting materials, (OH)2 SiPc, (OH)2 GePc and (OH)2 SnPc,
were prepared, purified and characterised according to literature procedures.19 ZnPc and ClAlPc complexes were obtained
from Sigma-Aldrich. The MPcSmix complexes are known1
to contain a mixture of the di-, tri-, and tetra-sulfonated
derivatives with an average of three sulfonate groups per
molecule (Fig. 1). High pressure liquid chromatography was
employed to characterize the mixture. BSA was purchased from
FLUKA and phosphate buffer solution (PBS, pH 7.4) was used
as solvent.
Fig. 1
Molecular structure of MPcSmix . M = Al, Si, Zn, Ge or Sn.
Equipment
Absorption spectra were recorded on a Varian Cary 500 UV-VisNIR spectrophotometer. Fluorescence excitation and emission
spectra were recorded on a Varian Eclipse spectrofluoremeter.
Light intensities were measured with a POWER MAX5100
(Molelectron detector incorporated) power meter. Triplet lifetimes and yields were recorded on a flash photolysis system,
with excitation pulses generated by a Nd:YAG laser (QuantaRay, 1.5 J/8 ns)-pumped dye laser (Lambda Physic FL 3002,
This journal is
© The Royal Society of Chemistry and Owner Societies 2005
Pyridine 1 dye in methanol). The analyzing beam source was
from a Thermo Oriel xenon arc lamp, and photomultiplier tube
was used as a detector. Signals were recorded with a two-channel
digital real-time oscilloscope (Tektronix TDS 360). High pressure liquid chromatography (HPLC) was performed on a QuadGradient HPLC system, Agilent 1100 Series; fitted with an
analytical column, l Bondapak C18 (390 × 3.00 mm) and
connected to a variable wavelength UV-Vis detector (set at k =
365 nm). The mobile phase comprised of 50:50 methanol:water
mixture, with a flow rate of 1 ml min−1 and sample injection
volume of 20 ll.
Photodegradation methods
Photodegradation (U d ) quantum yield determinations were carried out using the experimental set-up and equations described
in detail elsewhere.20–22 Typically, a 2 ml solution of MPcSmix
(absorbance ∼1.0 at the irradiation wavelength, the Q band) was
irradiated using a General electric Quartz line lamp (300 W).
A 600 nm glass cut off filter (Schott) and a water filter were
used to filter off ultraviolet and infrared radiations respectively.
An interference filter (Intor, 670 nm with a band width of
20 nm) was additionally placed in the light path before the
sample. Light intensity used for the experiments was 4.82 ×
1016 photons s−1 cm−2 .
MPcSmix binding to bovine serum albumin
The binding of the MPcSmix complexes to BSA was studied by
spectrofluorometry at room temperature (25 ◦ C). A solution of
BSA in PBS 7.4 was titrated with increasing concentrations of
the respective MPcSmix solution. BSA was excited at 280 nm
and fluorescence recorded between 290 and 500 nm. The
steady diminution in BSA fluorescence intensity with increase in
MPcSmix concentration was noted and used in the determination
of the binding constants and the number of binding sites on
BSA, according to eqn. (4).28–30
(F0 − F)
= log Kb + n log [MPcS]
(4)
log
(F − F∞ )
where F 0 and F are the fluorescence intensities of BSA in
the absence and presence of MPcSmix respectively; F ∞ , the
fluorescence intensity of BSA saturated with MPcSmix ; K b , the
binding constant; n, the number of binding sites on a BSA
molecule;
and
[MPcSmix ], the concentration of MPcSmix . Plots
(F0 −F)
of log (F−F
against log[MPcSmix ] would provide the values of
∞)
n (from slope) and K b (from the intercept).
For photophysical and photochemical studies, a molar ratio
of 10 : 1 (BSA : MPcSmix , with [BSA] = 7.5 × 10−5 M) was used.
This ratio was used due to the abundance of proteins within
most biological systems.
Fluorescence quantum yields
Fluorescence quantum yields (U F ) were determined by the
comparative method23,24 [eqn. (1)],
FAStd g2
U F = U F (Std)
FStd Ag2Std
(1)
where F and F Std are the areas under the fluorescence curves
of the MPcSmix and the reference, respectively. A and AStd are
the absorbances of the sample and reference at the excitation
wavelength respectively, and g and gStd are the refractive indices
of solvents used for the sample (in water, g = 1.330) and
reference (in ether, g = 1.352), respectively. Chlorophyll a in ether
(U F = 0.32)25 was employed as a reference. Both the sample and
reference were excited at the same wavelength. The absorbance
of the solutions at the excitation wavelength ranged between
0.04 and 0.05.
Triplet lifetimes and quantum yields
The deaerated solutions of the respective MPcSmix complexes
were introduced into a 2 mm × 10 mm spectrophotometric
cell and irradiated at the Q band with the laser system
described above. Triplet quantum yields (U T ) of the MPcSmix
complexes were determined by the singlet depletion method.20
A comparative method20,26 using zinc phthalocyanines tetrasulfonate (ZnPcS4 ) as standard was employed for the calculations,
eqn. (2).
U
Sample
T
=U
Std
T
DASample
eStd
S
S
Std Sample
DAS eS
MPcSmix fluorescence lifetimes were determined by monitoring
the fluorescence intensity as a function of quencher concentration. Here, two sets of data were obtained from: (i)
quenching of BSA fluorescence by MPcSmix and (ii) quenching of
MPcSmix fluorescence by BSA. For (i), the quenching of BSA by
MPcSmix , the changes in BSA fluorescence intensity were related
to MPcSmix concentrations by the Stern–Volmer relationship
[eqn. (5)]:
F0BSA
= 1 + kBSA
SV [MPcSmix ]
F BSA
(5)
and kBSA
SV is given by eqn. (6):
BSA
kBSA
SV = kQ sF
(6)
and F BSA are the fluorescence intensities of BSA in the
where F BSA
0
absence and presence of MPcSmix respectively; kBSA
SV , the Stern–
Volmer quenching constant; kQ , the bimolecular quenching
BSA
is
constant; and sBSA
F , the fluorescence lifetime of BSA. sF
obtained
estimated to be 10−8 s,10 thus from the values kBSA
SV
F BSA
from the plots of F0BSA versus [MPcSmix ], the value of kQ may be
determined from eqn. (6). For quenching of MPcSmix by BSA,
similar eqns. (7) and (8) apply.
F0MPc
= 1 + kMPc
SV [BSA]
F MPc
(7)
(2)
and DAStd
are the changes in the singlet state
where DAsample
S
S
absorbance of the MPcSmix complex and ZnPcS4 , respectively.
and eStd
are the singlet state extinction coefficients for the
esample
S
S
is the triplet
MPcSmix complex and ZnPcS4 , respectively. U Std
T
quantum yield for the standard ZnPcS4 in aqueous solution
(U T = 0.56).27
Quantum yields of internal conversion (U IC ) were obtained
from eqn. (3), which assumes that only three processes (fluorescence, intersystem crossing and internal conversion), jointly
deactivate the excited singlet state of an MPcSmix molecule.
U IC = 1 − (U F + U T )
Determination of MPcSmix fluorescence lifetimes
(3)
MPc
kMPc
SV = kQ sF
MPc
0
(8)
MPc
where F
and F
are the fluorescence intensities of MPcSmix
in the absence and presence of BSA respectively; kMPc
SV , the Stern–
Volmer quenching constant; kQ , the bimolecular quenching
constant; and sMPc
F , the fluorescence lifetime of MPcSmix . In both
cases, kQ is assumed to be the same. Using kMPc
SV from the plot
generated by eqn. (7), and kQ from eqn. (6), we can obtain the
from eqn. (8).
value of sMPc
F
The determination of fluorescence lifetimes afforded the
calculation of rate constants for processes deactivating the
excited singlet state, since the quantum yields are known. Values
for the rate constants for fluorescence (kF ), internal conversion
Photochem. Photobiol. Sci., 2005, 4, 510–516
511
(kIC ) and intersystem crossing (kISC ) were obtained using eqns.
(9a)–(9c):
kF =
UF
sF
(9a)
kIC =
U IC
sF
(9b)
kISC =
UT
sF
(9c)
Results and discussion
Effects of BSA binding on photophysical and photochemical
properties of MPcSmix complexes
Fig. 2 shows the ground state electronic absorption spectra of
MPcSmix mixtures in PBS 7.4. The Soret bands (not shown in
Fig. 2) were observed between 339 and 349 nm for the MPcSmix
complexes. The spectra of these complexes was the same in
unbuffered water, that is in the absence of ions present in buffered
solutions. Thus the ions in the buffered solution did not affect
the aggregation behaviour of these complexes. AlPcSmix , SiPcSmix
and GePcSmix are monomeric in nature, while ZnPcSmix and
SnPcSmix are aggregated. These observations were recently31,32
confirmed by the addition of a surfactant, Triton X-100 to these
complexes. AlPcSmix , SiPcSmix and GePcSmix gave no change in
spectra, confirming lack of aggregation whereas ZnPcSmix and
SnPcSmix gave marked enhancements of Q band intensity and
a corresponding collapse of the peak around 630 nm, which is
believed to be due to dimeric species. Aggregation in sulfonated
MPc complexes is characterised by the presence of two main
bands (instead of one for monomeric species) in the visible
region. The lower energy band is due to the monomer and the
higher energy band due to the aggregated species. The spectra
for SnPcSmix and ZnPcSmix show more monomerization at the
lower concentrations used in Fig. 2b. The presence of multiple
peaks in the Q band region of the un-aggregated SiPcSmix (shown
in Fig. 2) and GePcSmix complexes, could be an indication
of the presence of differently substituted components in the
mixture. The degree of aggregation increases with lipophilicity,
hence the prevalence of the less sulfonated fractions in solution
is expected to increase aggregation. The prevalence of less
sulfonated fractions in the aggregated ZnPcSmix and SnPcSmix
complexes and more sulfonated fractions in GePcSmix , AlPcSmix
and SiPcSmix was confirmed by HPLC, Figs. 3 and 4. From
HPLC, it is expected that the most highly sulfonated (most
soluble) would be the first to be eluted from the chromatographic
column, and so give the lowest retention time and that the less
sulfonated fractions as expected, give the highest retention times.
Thus the HPLC signals with the lowest retention times (∼1 min)
are assigned to the tetrasulfonated fractions using tetrasulfozinc
phthalocyanine (ZnTSPc) as reference. ZnPcSmix and SnPcSmix
gave similar traces (Fig. 3), with appreciable signals in the
relatively high retention time regions of the HPLC traces,
whereas GePcSmix AlPcSmix and SiPcSmix showed signals at low
retention times. These observations confirm the prevalence of the
highly sulfonated fractions in AlPcSmix , SiPcSmix and GePcSmix ,
hence less aggregation, while in ZnPcSmix and SnPcSmix , the less
sulfonated fractions are prevalent, and are more aggregated.
Fig. 3 HPLC trace for GePcSmix .
Fig. 4
Fig. 2 Ground state electronic absorption spectra of MPcSmix mixtures
in (a) PBS 7.4 (concentrations: ∼5 × 10−6 M for AlPcSmix and SiPcSmix ;
1.2 × 10−5 M for ZnPcSmix and SnPcSmix ) and (b) unbuffered water
(concentrations: ∼4 × 10−7 M for AlPcSmix and SiPcSmix; 8 × 10−7 M
for ZnPcSmix and SnPcSmix ).
512
Photochem. Photobiol. Sci., 2005, 4, 510–516
HPLC trace for ZnPcSmix .
Addition of BSA to solutions of the MPcSmix species (Fig. 5)
gave the same results as Triton X-100 addition, further confirming the aggregated nature of ZnPcSmix and SnPcSmix . The
monomerization effect of serum albumin on photosensitizers is
well documented.33–35 For the un-aggregated AlPcSmix complex,
Fig. 5 UV/Vis absorption spectral change observed on addition of
BSA to ZnPcSmix in PBS 7.4 (concentrations: ZnPcSmix = 1.63 × 10−6 M
and BSA = 1.63 × 10−5 M).
Fig. 7 Ground state electronic absorption (i) and fluorescence emission
(ii) spectra of BSA in PBS 7.4 (concentration = 2 × 10−5 M).
a small bathochromic shift (∼2 nm) in the Q band position
was observed on addition BSA, Fig. 6. This implies that a
complex is actually being formed between the photosensitizer
and the biopolymer. The lack of broadening or splitting in the
Q band, suggests that upon conjugation with BSA, AlPcSmix is
still not aggregated. A similar subtle change in Q band position
was observed for SiPcSmix , but not for GePcSmix , ZnPcSmix and
SnPcSmix . However some BSA–MPc conjugates are known not
to show shifts in the Q band compared to MPc complex alone.13
The electronic absorption and fluorescence spectra of BSA in
PBS 7.4 are shown in Fig. 7. BSA shows a strong absorption
around 280 nm (log e = 4.65) which is due to tryptophan and
tyrosine residues.4,36
Fig. 8 Fluorescence emission spectra of MPcSmix in the presence and
absence of BSA in PBS 7.4 (concentrations: AlPcSmix and ZnPcSmix =
3 × 10−6 M; BSA = 7.5 × 10−5 M).
Fig. 6 UV/Vis spectral changes observed on addition of BSA to
AlPcSmix in PBS 7.4. (concentrations: AlPcSmix = 7.5 × 10−6 M and
BSA = 7.5 × 10−5 M).
Fig. 8 shows the fluorescence emission spectra of AlPcSmix
and ZnPcSmix (free and BSA-bound). For the un-aggregated
species (AlPcSmix , SiPcSmix and GePcSmix ), BSA binding resulted
in reasonable fluorescence quenching which is manifested in decrease in the fluorescence intensity (shown in Fig. 8 for AlPcSmix )
and in fluorescence quantum yield (U F ) values (Table 1).
On the other hand, BSA binding resulted in increase in
fluorescence intensity (Fig. 8) and in U F values for the aggregated
ZnPcSmix and SnPcSmix , which further substantiates the fact that
BSA monomerizes the aggregated species, hence the increase in
fluorescence intensity rather than quenching. All the MPcSmix
complexes quenched BSA as evident from the decrease in BSA
fluorescence on titration with MPcSmix .
MPc photodegradation is believed to be a singlet oxygenmediated process,37 thus its efficiency should be related to the rate
of singlet oxygen generation, among other factors. Aggregation
is also known31 to reduce photostability, and Table 1 shows
that aggregated complexes, ZnPcSmix and SnPcSmix , show larger
degradation quantum yields. With the exception of GePcSmix for
which the value did not change (Table 1), MPcSmix photobleaching quantum yield (U d ) increased in the presence of BSA. We
attribute this increase to the formation of active oxidative
albumin species which could additionally photodegrade the
MPcSmix complex.
Table 1 Photophysical and photochemical parameters of MPcSmix complexes in PBS 7.4. Values in brackets are in the presence of 10 molar
proportions of BSA. [BSA] = 7.5 × 10−5 M
AlPcSmix
SiPcSmix
GePcSmix
ZnPcSmix
SnPcSmix
kQ /nm
kF /nm
UF
sT /ls
UT
U IC
105 /U d
UD
674
(677)
678
(679)
680
(680)
673
(676)
688
(685)
677
(683)
682
(682)
686
(686)
677
(681)
699
(688)
0.44V
(0.34)
0.34
(0.86)
0.30
(0.24)
0.16
(0.20)
0.05
(0.07)
2.93
0.44
0.12
0.42
2.90
0.45
0.21
2.76
0.68
0.03
2.95
0.53
0.31
2.52
0.59
0.36
0.40
(0.59)
0.71
(0.30)
0.45
(0.44)
3.65
(17.1)
1.59
(1.77)
U OP
U SnPcS
OP
(0.59)
0.49
(0.40)
0.68
(0.57)
0.45
(0.37)
0.42
(1.00)
Photochem. Photobiol. Sci., 2005, 4, 510–516
513
Photosensitized oxidation of BSA
MPcSmix photodegradation within the BSA–MPcSmix conjugate,
was accompanied by substantial increase in intensity in the
UV region of the spectrum (Fig. 9), implying that BSA
photodegrades in the presence of singlet oxygen, generating
products which absorb at about the same region as the original
BSA absorbing components, hence the increase in the intensity
in the UV region. Thus, BSA competes with the MPcSmix for
singlet oxygen. It is difficult to identify exactly which species is
responsible for the increase in intensity in the UV region. Due to
the relatively large concentration of BSA used in the experiments
and the high rate constants for reaction of singlet oxygen with
some amino acid side chains,38 it is logical to think that BSA
would be a target for singlet oxygen. A series of endoperoxides
and hydroperoxides have been indicated as products of amino
acid side chain oxidations in proteins.38 In order to quantify
the efficiency of BSA photooxidation, the increase in intensity
in the UV region (at 305 or 330 nm) was used, and related to
the photooxidation quantum yield (F OP ), by the usual eqn. (10)
employed for photodegradation studies:
U OP =
(At − A0 )V/e
Iabs t
(10)
where At and A0 are the absorbances at 305 or 300 nm (both
wavelength gave the same results) after irradiation for t s and
before irradiation respectively; V , the reaction volume; e, the
molar extinction coefficient of oxidation product; and I abs , the
intensity of absorbed light in photon mol s−1 .
Fig. 9 Spectral changes observed on photolysis of AlPcSmix in the
presence of BSA. pH = 7.4. Irradiation wavelength at AlPcSmix
complexes Q band maximum (674 nm). Light intensity used was 4.82 ×
1016 photons s−1 cm−1 (concentrations: AlPcSmix = 6 × 10−6 M and
BSA = 6 × 10−5 M). Irradiation time interval = 1 min.
Fig. 10 Determination of MPcSmix –BSA binding constant in PBS 7.4
(BSA concentration = 2 × 10−5 M).
Since the identity of the new species absorbing in the UV
region is not known (hence the extinction coefficient is not
known), eqn. (10) may not be used directly. We employed a
514
Photochem. Photobiol. Sci., 2005, 4, 510–516
relative method described below. The rate of formation (R) of
the oxidation products is given by eqn. (11):
R=
(At − A0 )V/e
t
(11)
Combining eqns. (10) and (11) gives eqn. (12a):
U OP =
R
Iabs
(12a)
For another photosensitizer, we can write a similar eqn. (12b):
U ′OP =
R′
′
Iabs
(12b)
Taking the ratio of eqn. (12b) over (12a), gives eqn. (13):
R′ Iabs
U ′OP
=
′
U OP
RIabs
(13)
Values of BSA photooxidation quantum yields were obtained
relative to that in SnPcSmix , which is given an arbitrary value
of 1.00. The relative values of FOP for all MPcSmix complexes
studied are listed in Table 1. Higher values were observed for Al,
Ge, and Sn complexes.
Interaction of MPcSmix with BSA
The binding constants (K b ) obtained for MPcSmix binding to
BSA, together with the binding stoichiometry of the complex
formed were obtained using eqn. (4) (Fig. 10, for SiPcSmix ); and
the results are presented in Table 2 for [BSA] = 2.0 × 10−5 M and
[MPc] ranging from 1.58 × 10−6 to 1.58 × 10−5 M. In addition
for the aggregated SnPcSmix and ZnPcSmix lower concentrations
([BSA] = 3.3 × 10−7 M and [MPc] = 1.67 × 10−7 to 8.0 × 10−7 M)
were employed. The binding contant values were the same when
unbuffered water was employed. Again showing that the salts in
the buffered solutions do not affect the aggregation behaviour of
these complexes. The values for the unaggregated Ge, Si and Al
complexes are typical of MPc–albumin interactions in aqueous
solutions,28 and did not change on dilution to low concentrations
(∼10−7 M) similar to those employed for SnPcSmix and ZnPcSmix
complexes (for binding and quenching constants shown in
brackets, Table 2). The highest value of K b was obtained for
AlPcSmix which is particularly monomeric, followed by GePcSmix
and SiPcSmix . The aggregated SnPcSmix and ZnPcSmix complexes
gave the lowest K b values, but these values increased considerably
(∼three-fold) at low concentrations as shown in Table 2. This
shows that aggregation plays an important role in the binding.
The involvement of a MPc dimer in BSA binding is indirect,
via dissociation into monomers. Consequently, K b values for
dimeric species are expected to depend largely on, and be limited
by the inherent dimer dissociation constant. The variation of
K b with the nature of the complex in Table 2 could also be
a reflection of the relative affinity of BSA for the respective
MPcSmix species. Since it is known that serum albumins have
high affinities for negatively charged molecules,39 the observed
larger values of K b for AlPcSmix , SiPcSmix and GePcSmix is a result
of the predominance of highly sulfonated derivatives as shown
by HPLC (Fig. 3). The number of binding sites on BSA (n)
obtained from the experiments is ∼1 (Table 2), which suggests a
1 : 1 stoichiometry for all the MPcSmix –BSA conjugates.
Fluorescence quenching analysis
As discussed above, BSA and each of the MPcSmix species
display mutual quenching on one another. These reciprocative
fluorescence quenching could have arisen from either dynamic
(no complexation) or static (due to non-fluorescent ground state
Table 2 Quenching and binding data for MPcSmix complexes in PBS 7.4. Unless otherwise stated: [BSA] = 2.0 × 10−5 M and [MPc] ranges from
1.58 × 10−6 to 1.58 × 10−5 M. Values in brackets are for [BSA] = 3.3 × 10−7 M and [MPc] ranging from 1.67 × 10−7 to 8.0 × 10−7 M
AlPcSmix
SiPcSmix
GePcSmix
ZnPcSmix
SnPcSmix
a
K b /10−6 M−1
n
−4
kBSA
M−1
SV /10
−3
kMPc
M−1
SV /10
kQ a /10−12 M−1 s−1
sF /ns
kF /10−7 s−1
kISC /10−7 s−1
kIC /10−7 s−1
17.21
1.29
0.81
0.10 (0.34)
0.08 (0.21)
1.4
1.3
1.1
1.0
1.0
11.45
6.90
4.54
7.36 (6.72)
1.98 (3.19)
58.43
34.36
18.48
11.40
6.90
4.54
7.36 (6.72)
1.98 (3.19)
5.13
4.98
4.07
2.927
8.58
6.83
7.37
5.52
8.58
9.04
16.71
18.28
2.34
4.22
0.74
10.69
Values determined from BSA quenching by MPcSmix .
complex) quenching. The Stern–Volmer analysis of fluorescence
data was used to discern the actual quenching mechanism.
Figs. 11 and 12 show the quenching of BSA and SiPcSmix in PBS
7.4, respectively by each other. The slopes of the plots shown
and kMPc
as inserts gave kBSA
SV
SV , respectively. Fig. 12 shows the
quenching behaviour of MPcSmix typical of the un-aggregated
complexes (Al, Si and Ge complexes). As discussed above for
the aggregated Zn and Sn complexes, there was an increase in
fluorescence intensity (rather than quenching) upon addition of
BSA, hence the kMPc
SV values could not be determined, whereas all
MPcSmix complexes could quench BSA with kBSA
SV values shown
in Table 2.
Fig. 11 Fluorescence emission spectral changes for BSA on addition
of increasing concentrations of SiPcSmix . Inset: Stern–Volmer plot for
SiPcSmix quenching of BSA. (Concentrations: BSA = 2 × 10−5 M;
SiPcSmix varies from 1.67 × 10−6 M to 1.5 × 10−5 M.)
Fig. 12 Fluorescence emission spectral changes of GePcSmix on addition of increasing concentrations of BSA. Inset: Stern–Volmer plot for
BSA quenching of GePcSmix . (Concentrations: GePcSmix = 4 × 10−6 M;
BSA varies from 1.4 × 10−5 M to 5.7 × 10−5 M.)
Values of kQ were determined from eqn. (6) for quenching
of BSA by MPc and are listed in Table 2. These values
are of the order of 1012 M−1 s−1 , Table 2. The acceptable
value of kQ for dynamic quenching according to Einstein–
Smoluchowski approximation40 at room temperature is of the
order of 1010 M−1 s−1 . The high values of kQ observed in Table 2,
suggests that the fluorescence quenching of BSA by MPcSmix is
not initiated by dynamic quenching, but by static quenching,
and kMPc
values in eqns. (6) and (8) are due to
and the kBSA
SV
SV
static quenching. These static quenching constants (kBSA
and
SV
kMPc
SV ; Table 2) for BSA and the MPcSmix complexes reveal the
relative degrees of interaction between BSA and the MPcSmix
complexes. Both constants are highest for AlPcSmix . From these
values, it could be inferred that AlPcSmix possesses the highest
degree of interaction with BSA while SnPcSmix has the lowest.
for ZnPcSmix and SnPcSmix
The observed lower values of kBSA
SV
are not unconnected with their aggregation, and the relatively
higher values for the former could imply the lower aggregation
tendency of ZnPcSmix compared to SnPcSmix . The high kBSA
SV
and kMPc
SV values for AlPcSmix compared to the other monomeric
complexes (SiPcSmix and GePcSmix ), suggests that the latter two
have lower affinities for BSA than the former.
Fluorescence lifetimes of the MPcSmix complexes (sMPc
F , Table 2) were calculated from kMPc
SV and kQ values using eqn. (8),
and the values are quite close to the typical MPc fluorescence
lifetimes.19,28 The procedure described here for calculation of sMPc
F
could however not be employed in the case of the aggregated
ZnPcSmix and SnPcSmix , whose fluorescence intensities increased
on BSA addition, hence the kBSA
SV values could not be calculated.
Among the three: AlPcSmix , SiPcSmix and GePcSmix which are
monomeric, fluorescence lifetime decreases as the atomic mass
of central metal ion increases, most probably due to an enhanced
intersystem crossing. The literature value for ZnPcSmix 27 is
notably less than those calculated for AlPcSmix , SiPcSmix and
GePcSmix (Table 2) which is attributable to the aggregated nature
of the complex.
Kinetic data
The rate constants for the excited singlet state deactivation
processes (kF , kIC and kISC ) were calculated [using eqn. (9a)–(9c)]
and are listed in Table 2. The values could not be obtained for
SnPcSmix whose sF value was not available; but among the three
unaggregated species, AlPcSmix showed the highest values of kF ,
while GePcSmix showed the lowest. Comparison of ZnPcSmix
with the rest is not appropriate due to its aggregated nature.
The values of kISC , as expected, increased with the mass of the
central metal ion, which is a manifestation of the heavy atom
effect that promotes intersystem crossing. As expected, ZnPcSmix
gave the highest value of kIC , which is again readily attributed
to aggregation. GePcSmix gave the lowest value of kIC , indicating
that this species is perhaps the most photoactive in the list. The
superior photoactivity of this species is also shown in Table 1,
where its values of U F , U T and U D are consistently high.
Conclusions
In conclusion, this work provides a qualitative and quantitative
interpretation of the interaction of MPcSmix mixtures with
BSA. Results of binding experiments showed that each of the
MPcSmix mixtures formed a 1 : 1 adduct with BSA, but the
binding feasibilities varied markedly, as evident from the values
of binding constants (K b ) for the MPcSmix –BSA adduct; with
AlPcSmix giving the highest value and SnPcSmix giving the lowest.
The observed trend in the values of K b was explained in terms of
aggregation and the greater affinities of some of the species for
BSA than others. The spectral as well as photophysicochemical
Photochem. Photobiol. Sci., 2005, 4, 510–516
515
properties of the mixtures were altered in the presence of
BSA. This work also provides an approximate but simple route
to the determination of fluorescence lifetimes of MPc complexes,
using steady-state measurements.
19
Acknowledgements
This work has been supported by the National Research
Foundation of South Africa as well as Rhodes University.
AO thanks ICSC world laboratory and Mellon foundation for
scholarships.
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