Aggregation of two carboxylic derivatives of porphyrin and their affinity to bovine serum albumin

Article abstract of DOI:10.1016/j.saa.2005.09.012

Aggregation of two porphyrin derivatives with carboxylic groups, 4-oxo-4-((4-(10,15,20-triphenyl-21H,23H-porphin-5-yl)phenyl)amino)butanoic acid (MAC) and 4,4′,4″,4?-[21H,23H-porphine-5,10,15,20-tetrayltetrakis(4,1-phenyleneimino)]tetrakis(4-oxo-butanoic acid) (TA4C), and their affinity to bovine serum albumin were investigated via absorption spectrometry, ¹H NMR and fluorescence spectrometry. MAC and its complexes with β-cyclodextrin could form aggregates in an aqueous solution while TA4C was self-associated loosely. From the absorbance profiles of MAC in the titration of bovine serum albumin, hypochromicity was observed without any shift of the maximum absorbance wavelength. In both absorption spectra of TA4C in aqueous solutions and in solid state, three Q bands appeared in the visible region. In the measurements of absorption and fluorescence spectra upon titration of BSA, some spectral changes of TA4C were observed. The whole procedure of titration could be divided into three successive stages. The three-banded profiles of TA4C might be explained according to a loose dimer model.

Full text of DOI:10.1016/j.saa.2005.09.012

Spectrochimica Acta Part A 64 (2006) 1032–1038
Aggregation of two carboxylic derivatives of porphyrin
and their affinity to bovine serum albumin
Yao-Bing Yin, Yi-Nong Wang, Jian-Biao Ma∗
Key Laboratory of Functional Polymer Materials (Ministry of Education), Nankai University, Tianjin 300071, PR China
Received 18 July 2005; received in revised form 12 September 2005; accepted 13 September 2005
Abstract
Aggregation of two porphyrin derivatives with carboxylic groups, 4-oxo-4-((4-(10,15,20-triphenyl-21H,23H-porphin-5-yl)phenyl)amino)-
butanoic acid (MAC) and 4,4ꢀ,4ꢀꢀ,4ꢀꢀꢀ-[21H,23H-porphine-5,10,15,20-tetrayltetrakis(4,1-phenyleneimino)]tetrakis(4-oxo-butanoic acid) (TA4C),
and their affinity to bovine serum albumin were investigated via absorption spectrometry, 1H NMR and fluorescence spectrometry. MAC and its
complexes with ␤-cyclodextrin could form aggregates in an aqueous solution while TA4C was self-associated loosely. From the absorbance profiles
of MAC in the titration of bovine serum albumin, hypochromicity was observed without any shift of the maximum absorbance wavelength. In both
absorption spectra of TA4C in aqueous solutions and in solid state, three Q bands appeared in the visible region. In the measurements of absorption
and fluorescence spectra upon titration of BSA, some spectral changes of TA4C were observed. The whole procedure of titration could be divided
into three successive stages. The three-banded profiles of TA4C might be explained according to a loose dimer model.
© 2005 Elsevier B.V. All rights reserved.
Keywords: Aggregation; Binding modes; Three-banded Q bands; Fluorescence; Absorption spectrum
1. Introduction
ence of substituents, monoprotonation and steric hindrance
on the three-banded phenomena was observed. The three-
banded Q bands existed in the UV–vis spectra of some mono-
protonated porphyrins [15], 5,15-di(4-hydrocyphenyl)-10,20-
di(4-hexadecyloxyphenyl)porphyrin [16], five tri-(N-methyl-4-
pyirdiniumyl) porphyrins with an amino acid or peptide side-
chain on the fourth meso aromatic substituent [17] and four
protoporphyrin IX derivatives with 2-aminoglycosamide group
[18] in aqueous media. In the absorption spectrum of tetra-
(p-N,N-diphenylamino)phenylporphine (TDPAPPH2) in chlo-
roform solution, three Q bands also occurred due to non-planar
distortion of the porphine ring originated from big steric hin-
drance [19]. Unfortunately, the nature and the significance of
three-banded profile of different porphyrins are not clear up to
date.
In the present study, an attempt is to gain more insight into
the aggregation behavior of some porphyrins and their inter-
action with biological molecules. Two porphyrin derivatives
with butanoic acid groups, 4-oxo-4-((4-(10,15,20-triphenyl-
21H,23H-porphin-5-yl)phenyl)amino)butanoic acid (MAC) and
4,4ꢀ,4ꢀꢀ,4ꢀꢀꢀ-[21H,23H-porphine-5,10,15,20-tetrayltetrakis(4,1-
phenyleneimino)]tetrakis(4-oxo-butanoic acid) (TA4C), as
illustrated in Chart 1, were studied via absorption spectrom-
etry, 1H NMR and fluorescence spectrometry. The effect of
Aggregation of porphyrin derivatives is known to play an
important role in biological events, such as photosynthetic light
energy conversion, oxygen transport, and biological catalysis.
Their affinity to biological molecules is often involved in the
transportation and metabolism of porphyrins in human bodies.
It is found that the formation of porphyrin aggregates in aqueous
solutions is often affected by the chemical structure of por-
phyrins, ionic strength, temperature, pH and surfactants [1]. The
affinity of some porphyrins to bovine serum albumin or human
serum albumin was studied [2–6]. Especially, much attention
was paid on protoporphyrin IX [7], deuteroporphyrin [8] and
other carboxylic porphyrins [9,10] in the past decades. More-
over, some cyclodextrin derivatives could bind water-soluble
porphyrins [11,12] so as to preclude the porphyrin–porphyrin
aggregation [13].
On the other hand, three Q bands as an interesting spec-
tral characteristic of tetracarboxyphenylporphine (TCPP) in an
aqueous solution occurred in the visible region [14]. The influ-
∗
Corresponding author. Tel.: +86 22 23507746; fax: +86 22 23502749.
E-mail address: jbma@nankai.edu.cn (J.-B. Ma).
1386-1425/$ – see front matter © 2005 Elsevier B.V. All rights reserved.
doi:10.1016/j.saa.2005.09.012

Y.-B. Yin et al. / Spectrochimica Acta Part A 64 (2006) 1032–1038
1033
spectra were measured using JY FluoroMax-P spectrofluorom-
eter after the samples were equilibrated for 1 h.
2.3. Spectra titration
A stock solution was prepared by dissolving TA4C in phos-
phate buffer at pH 7.4. A stock solution of MAC was prepared
by successive dissolution of MAC into THF, addition of distilled
water, and removement of THF via evaporation in vacuo. The
quartz cuvettes were cleaned in 3 mol dm−3 nitric acid overnight
and washed with distilled water, ethanol, distilled water, and the
sample solution before any measurement. Both the porphyrins
fluorescenceandabsorbanceweretitratedwithproteinsolutions.
All experiments were carried out at pH 7.4 in phosphate buffer
containing 0.01 mol dm−3 NaCl at 25 ◦C. The concentration of
porphyrin derivatives maintains 1 × 10−6 mol dm−3
.
3. Results and discussion
3.1. Beer’s law
Chart 1. Structures of two porphyrin derivatives with carboxylic groups.
The Beer’s law titrations of the TA4C at pH 7.4 (Fig. 1)
and MAC at pH 7.4 in the presence and the absence of ␤-CD
were performed respectively at a suitable wavelength. As seen in
Fig. 1, the photophysical properties of TA4C poorly adhere to the
Beer’s law in the present case, indicating that molecules of TA4C
form aggregates even in a dilute aqueous solution. Remarkable
deviation from the Beer’s law is observed for MAC both in the
presence and the absence of ␤-CD (plots not shown). In fact, it
is difficult to measure the spectral absorption of MAC because
it is per se insoluble in distilled water. Although ␤-cyclodextrin
as protective molecules can preclude the porphyrin–porphyrin
interaction [13], deviation from the Beer’s law is significant
for MAC in the presence of ␤-CD. The result implies that the
tetraphenylporphyrin with only one butanoic acid group pre-
dominantly trends to aggregate in aqueous media owing to its
poor hydrophilicity. It is in line with the previous results that the
increased lipophilicity of porphyrins with long nonpolar alkyl
␤-cyclodextrin on aggregation of MAC in an aqueous medium
was investigated. The spectral characteristic of the three Q
bands was observed and discussed to elucidate the aggregation
of TA4C in an aqueous solution and the binding of TA4C to
BSA.
2. Experimental
2.1. Materials
Monomethoxy poly(ethylene glycol) was supplied by Sigma
Chemical Company. Bovine serum albumin (BSA) was pur-
chased from Institute of Hematology, Chinese Academy of
Medical Sciences. All chemicals were of analytic grade and
purified prior to use. 5,10,15,20-tetrakisphenylporphyrin (TPP)
was prepared according to the previous literature. 5,10,15,20-
Tetrakis(4-aminophenyl)porphyrin (p-TAPP) [20] and 5-(4-
aminophenyl)-10,15,20-triphenylporphyrin (p-MAPP) [21,22]
were prepared from the corresponding nitro-substituted ben-
zaldehyde and pyrrole [23] via the condensation and suc-
cessive reduction with SnCl2 in 12 mol dm−3 HCl–acetic
acid (1:1, v/v) at 65 ◦C. TA4C and MAC were synthe-
sized via esterification of p-TAPP and p-MAPP with succinic
anhydride.
2.2. Instruments
Proton NMR spectra were measured on a Varian UNITY
Plus-400 spectrometer. Reflective solid UV–vis spectra
were recorded with a JASCO U-570 spectrophotometer.
Ultraviolet–visible (UV–vis) spectra were obtained on a SHI-
MADZUUV-2101PCspectrophotometer wherethe temperature
of the solutions was maintained by the use of a constant-
temperature circulation pump (Tulabo F12) and a variable-
temperature cell holder (Hitachi). Steady-state fluorescence
Fig. 1. Beer’s law titration of TA4C.

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Y.-B. Yin et al. / Spectrochimica Acta Part A 64 (2006) 1032–1038
Fig. 3. Representative profile of 1H NMR of TA4C: (a) in DMSO-d6; (b)
1 × 10−5 mol dm−3 in D2O.
ratioofporphine/ethylenedecreasesdramaticallyto0.05atroom
temperature (Fig. 3b). 1H NMR spectrum of TA4C at a higher
concentration give only the ethylene peak at 2.70–2.80 ppm and
the proton peaks of the porphyrin residues can not be observed
even though the solution of TA4C is green and exhibits the char-
acteristic porphyrin bands in its electronic spectrum (1H NMR
spectrum not shown). This phenomenon suggests that TA4C do
not present as free molecules in an aqueous solution at a higher
concentration, which is in good agreement with the recent study
on porphyrinic polymer in another group [26].
Fig. 2. Absorption spectra of MAC. (a) MAC in THF (solid line); (b) MAC-␤-
CD in water (dash line); (c) MAC in water (dot line); The inset is the magnified
Q bands of MAC.
chain would result in a pronounced tendency toward the aggre-
gation [24].
3.2. Aggregates
The number of butanoic acid groups influences aggregation
of MAC and TA4C in solid state. Their reflective UV–vis spec-
tra are displayed in Fig. 4. It can be seen that the spectral profile
of MAC is similar to that of TPP, but the spectral profile of
TA4C is distinctly different from that of MAC. The absorption
band of MAC and TPP are 374, 523, 560, 600, 657 nm and 353,
509, 552, 592, 650 nm, respectively. A butanoic acid group ren-
ders the reflective peaks of MAC red shift. Soret bands of MAC
and TPP occur at shorter wavelengths (374 and 353 nm, respec-
tively). This indicates that molecules of MAC and TPP exist in
aggregates in solid state. In contrast with this result, the Soret
band of TA4C splits into two peaks at 431 and 467 nm because
TA4C exist in the monomolecular state and aggregates. The Q
bands of TA4C precipitated from an aqueous solution surpris-
The UV–vis absorption spectra of MAC in tetrahydrofuran
solution and in an aqueous solution in the presence and the
absence of ␤-cyclodextrin are displayed in Fig. 2. It can be
clearly seen that the absorbance intensities of MAC in the aque-
ous solution are lower for the Soret band and higher or equal
for Q bands than those in the organic solution, respectively. The
result suggests that intensity transfer from the B- to the Q-band
region occurs owing to an excitonic coupling between B and
Q transition [25]. Moreover, a blue-shifted shoulder appears at
403 nm for MAC both in the organic solution and in the aqueous
solution in the presence of ␤-cyclodextrin, while the absorption
of Q-bands in the visible region exhibits a slight red shift. These
spectral profiles indicate the formation of porphyrin aggregates.
In comparison with the data in the absence of ␤-cyclodextrin,
the corresponding bands exhibit red shift and slightly higher
intensities after MAC interacts with ␤-cyclodextrin. This result
is attributed to the inclusion complexes of the porphyrin with
␤-cyclodextrin, which is consistent with the previous studies
[11,12]. In addition, the absorption spectrum of this inclusion
complex is not changed after addition of equimolar BSA solu-
tion (absorption spectra not shown), suggesting that the complex
of MAC with ␤-cyclodextrin can not bind to BSA.
At a higher concentration of TA4C than 1 × 10−4 mol dm−3
in an aqueous solution, a remarkable deviation from the Beer’s
law was observed owing to the formation of TA4C aggre-
gates. Further evidence for the aggregates was observed in
the 1H NMR measurement of TA4C. 1H NMR spectra of
TA4C in DMSO-d6 and in phosphate buffer (pH 7.4) at a con-
centration of 1 × 10−5 mol dm−3 are shown in Fig. 3. In the
molecular solution of TA4C in DMSO-d6, the molar ratio of
porphine/ethylene, calculated from integration values, is 0.25
(Fig. 3a). At a lower concentration in the buffer, however, the
Fig. 4. Reflective UV–vis spectra of TPP (dot line), MAC (solid line) and TA4C
(dash line).

Y.-B. Yin et al. / Spectrochimica Acta Part A 64 (2006) 1032–1038
1035
Fig. 5. Absorption spectra of MAC upon titration with BSA in phosphate buffer
(pH 7.4). BSA concentration (in mol dm−3) increases in the order 0.0 (B),
0.02 × 10−6 (D), 0.05 × 10−6 (F), 0.1 × 10−6 (H), 0.15 × 10−6 (J), 0.2 × 10−6
(L), 0.3 × 10−6 (N), 0.5 × 10−6 (P), 1.0 × 10−6 (R) for curves B–R.
ingly show three peaks at 529, 571, and 665 nm, respectively.
A similar spectral profile in visible region also occurs for the
TA4C aqueous solution.
3.3. Interaction with BSA
It is difficult to carry out the titration of MAC with an aqueous
BSA solution since MAC is insoluble. A simple experiment on
the interaction of the water-insoluble porphyirn with BSA was
performed adopting an organic solvent evaporation method as
applied for preparing polymeric nanoparticles [27]. In order to
investigate interaction of MAC with BSA, a series of MAC solu-
tions were prepared through dissolving tetrahydrofuran solu-
tions of MAC in distilled water and then removing almost all of
organic solvent by evaporation. The yellow transparent suspen-
sions were obtained finally and the absorption spectra revealed
the formation of aggregates of MAC (absorption spectrum not
shown). These suspensions of MAC were employed to perform
the titration with BSA solution and the spectral changes of MAC
are showed in Fig. 5. It is seen that the Soret band exhibits
slight hyperchromicity without any obvious shift of the max-
imum absorbance wavelength. The shoulder peak at 394 nm
as the spectral adsorption of MAC aggregates becomes weaker
accompanied with hyperchromicity of the Soret band, suggest-
ing that the aggregates are broken partially after interaction with
protein.
In Fig. 6, it is observed that the Soret band and four Q bands of
TA4C change upon titration with gradually increasing concen-
trations of BSA. The most obvious change occurs in the Soret
region of the absorption spectrum, where the intense band at
417 nm undergoes a red shift of 8 nm upon addition of BSA.
No isosbestic point is observed, which indicates the presence
of more than one type of porphyrin–BSA complexes in equilib-
rium with free porphyrins. As also illustrated in Fig. 6, there are
three successive stages of the spectral change during the titra-
tion, and one near isosbestic point exists during the second stage
of the titration as follows. At the first stage, the initial porphyrin
Fig. 6. Absorption spectra of TA4C upon the titration of BSA. (A) Concentration
of TA4C is 1 × 10−6 mol dm−3 and the concentrations of BSA as arrowhead are
0, 0.02 × 10−6, 0.05 × 10−6, 0.1 × 10−6, 0.15 × 10−6, 0.2 × 10−6, 0.3 × 10−6
,
0.5 × 10−6, 1.0 ×10−6, 2.0 × 10−6 mol dm−3, respectively; (B) the same titra-
tion expressed in the forms of a plot of the absorbance (᭹) and a plot of
wavelength (ꢀ) of the Soret band vs. concentration of BSA, respectively. Equal-
ized for 60 min.
spectrum and spectra at very low BSA concentrations obviously
do not exhibit an isosbestic point. Remarkable hypochromicity
accompaniedbyaslightredshiftattheSoretbandisobserved. At
this stage, the behavior of BSA is similar to that of water-soluble
macromolecules such as polyethylene glycol (PEG). Absorption
spectra of TA4C upon addition of PEG (Mn = 5000) aqueous
solution show hypochromicity only under the same experimen-
tal condition (absorption spectra not shown). The second stage
shows a near isosbestic point at 421.8 nm in the spectra pro-
file. It can be clearly observed that addition of BSA solution
induces a significant red shift of the Soret band, and that the
Soret maxima displays slight hypochromicity then followed by
hyperchromic shift. At even higher BSA concentrations, a grad-
ual hyperchromicity of the Soret band can be observed during
the third stage. Even though a large excess of BSA is introduced
into the TA4C solution, the absorption spectral profile of the
porphyrin exhibits slight hyperchromicity instead of a red shift
of the Soret band.
Moreover, usual curve fitting analysis for the observed
absorbance changes during the second stage gives the asso-

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Y.-B. Yin et al. / Spectrochimica Acta Part A 64 (2006) 1032–1038
Fig. 8. Fluorescence emission spectra of TA4C upon the titration of BSA, mea-
sured at excitation wavelength λem = 422 nm. Concentration of BSA increases
along with the direction of the arrowhead (in mol dm−3): 0.0, 0.02 × 10−6
0.05 × 10−6, 0.1 × 10−6, 0.15 × 10−6, 0.2 × 10−6, 0.3 × 10−6, 0.5 × 10−6
1.0 × 10−6, and 2.0 × 10−6. Equalized for 60 min.
,
,
more slight blue shift, and the half-peak width becomes narrow.
This spectral change and changes occured in the mirror images
in fluorescence emission spectra, as mentioned in the following
section, exhibits that Qx(0,0) is also involved in the formation
of the three-banded profile. Significantly, this phenomenon is
not in line with the absorption spectral change of porphyrin-
containing poly(N-isopropylacrylamide) in an aqueous solution,
which displays bathochromic Qy(0,0) and Qy(1,0) bands so that
the Qy(0,0) band partially overlaps with Qx(1,0) band [28].
Fig. 8. exhibits the fluorescence emission spectra of TA4C
binding to BSA at different amount ratios measured at exci-
tation wavelength λem = 422 nm. As inferred from Fig. 8, the
fluorescence emission peaks of Q(0,1) significantly display three
profiles, one profile is that the fluorescence emission intensity
increasesgradually, theotheristhat thehalf-peakwidthbecomes
narrow, the third is that the Q(0,1) peak appears red shift at
the beginning and then blue shift. It can be also seen that the
fluorescence emission peaks of Q(0,0) exhibit similar character-
istics. These results imply that Q(0,1) and Q(0,0) peaks both
are involved in the interaction procedure between porphyrin
and BSA. It is known that the fluorescence emission spectrum
sometimes appears the mirror image of the absorption spectrum.
Taking together with the changes of the absorption spectra in the
titration procedure, it can be concluded that the Q(0,0) involved
in formation of the three-banded absorption spectra of TA4C in
an aqueous solution.
Fig. 7. Q bands of TA4C upon the titration of BSA. For curves a–j, BSA
concentration (in mol dm−3) was 0.0, 0.02 × 10−6, 0.05 × 10−6, 0.1 × 10−6
0.15 × 10−6, 0.2 × 10−6, 0.3 × 10−6, 0.5 × 10−6, 1.0 × 10−6, 2.0 × 10−6
respectively. Equalized for 60 min.
,
,
ciation constant to be 4.3 × 107 dm3 mol−1 at 298 K. The
affinity of TA4C to BSA is stronger than that of other car-
boxylic porphyrins and common tetraphenylporphyrin deriva-
tives (K = 104–106 dm3 mol−1). The reason may be that besides
␲–␲ stacking interaction, hydrophobicity, hydrogen bonds gen-
eratingfromcarboxylgroupsandlowaggregation, theadditional
hydrogen bonds generating from four amide bonds enhance the
ability of TA4C binding to BSA.
3.4. Changes of the Q bands during absorption and
fluorescence titration
In order to further probe characteristics of the three-banded
spectra of TA4C, we analyzed the absorption spectra in visible
region of TA4C upon the titration of BSA. The visible spectral
changes of the Q bands upon the titration of BSA are shown
in Fig. 7. It can be seen clearly that the number of Q bands
changesfrom3to4. Moreover, thespeciestransformationoccurs
at the second stage of the absorption spectral change during
the titration. These results evidence that the porphine ring is
involved in the interaction of porphyrin with protein. It also can
be seen that the blue shift of Qx(1,0) band, which is the major
change, leads to formation of three-banded spectrum while the
position of Qy(1,0) and Qy(0,0) bands remains almost unaltered.
Meanwhile, companying with the changes of Qx(1,0) band, the
Qx(0,0) band also undergoes a slight red shift followed by a
3.5. Aggregation mode of TA4C
As induced from Figs. 7 and 8, the blue shift of Qx(1,0) and
broadening of the half-peak width of Qx(0,0) are major changes
duringtheformationofthree-bandedspectrumofTA4C. Inother
words, the formation of three-peaked Q bands results from that
the blue-shifted Qx(1,0) merges with Qy(0,0), accompanying
withthattheblue-shiftedQx(0,0)becomesbroad, whichisinline
with the case of TCPP [14]. The “face to face” model has been
supported by a fluent of researches [29–32]. Based on our exper-

Y.-B. Yin et al. / Spectrochimica Acta Part A 64 (2006) 1032–1038
1037
Fig. 9. Loose dimer of TA4C in an aqueous solution.
iments and the abovementioned understanding of three-peaks Q
bands, we here propose a loose dimer model for association
behavior of TA4C in an aqueous solution (Fig. 9). According
to this loose dimer model, a face-to-face dimer of porphyrin
is formed. Two carboxylic groups are hydrogen-bonded to the
inner NH. Other six butanoic acid groups extend well-distributed
towards the environmental space. Scolaro et al. proposed a sim-
ilar hydrogen-bonding network but they were inclined to think
that it was not a better model [7]. On considering the simi-
lar spectral profiles of TA4C in solid state, the slight deviation
from the Beer’s law and the hydrophobic–hydrophilic interac-
tion, we think that the modified model could be more plausible
for the case of TA4C. In this case, the added salt could enhance
the hydrophobic–hydrophilic interaction and stabilize the aggre-
gated species [1]. The blue-shift of Qx(0,1) can be interpreted
as effects of hydrogen bonds on the conjugated system [33] of
porphine ring, and a twisted conformation for porphine ring due
to four carboxylic groups extending towards differently orienta-
tional space. Moreover, the loose dimer model can be utilized to
explain the three-banded spectrum of TA4C in solid state. After
precipitated from the aqueous solution, a loose dimer is stacked
with another similar unit via hydrogen bonds among butanoic
acid groups, leading to three-banded spectra (Fig. 4).
order to reach definitive conclusions concerning the nature of the
changes observed, a more detailed experimental examination of
these component transitions would be required.
4. Conclusions
In conclusion, the tight aggregates of MAC in an aqueous
solution lead to slight hypochromicity and the absence of obvi-
ousredshiftforMACuponthespectraltitrationofBSAsolution.
The complexes of MAC aggregates with ␤-cyclodextrin cannot
interact with BSA. The changes in the three-banded spectrum
of TA4C in visible region during the spectral titration with BSA
show that the titration of TA4C with BSA could be divided into
three stages. The three-banded profile of TA4C both in an aque-
ous solution and in solid state may be explained on the basis of
a loose dimer model.
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