CHARACTERIZATION OF WEAK ASSOCIATES BETWEEN
PORPHYRINS AND CONCANAVALIN A
Katarzyna Polska, Magdalena Makarska and Stanisław Radzki
Department of Inorganic Chemistry, Maria Curie-Skłodowska University
Pl. M.C. Skłodowskiej 2, 20-031 Lublin, Poland
e-mail: kmigut@hermes.umcs.lublin.pl
The interactions between water-soluble anionic and cationic porphyrins and Canavalia ensiformis lectin
- concanavalin A have been studied using spectrophotometric titration. It has been shown that concanavalin A
forms 1:1 molecular associates with all porphyrins studied. The calculated constants of association increase with
increasing pH. Potential application of such ion-pair complexes includes photodynamic therapy of cancer.
Keywords: porphyrins, complex Cu(II), concanavalin A, photosensitizer, photodynamic therapy (PDT)
1. INTRODUCTION
Binding of synthetic porphyrins to proteins such as human serum albumin,
immunoglubulin G and lipoproteins has been investigated by several groups [1-3]. These
systems are strongly connected with clinical application of porphyrins used as
photosensitizers in fluorescence detection and photodynamic therapy of cancer (PDT).
Porphyrins can accumulate in abnormal cells and, upon irradiation by light, destroy diseased
areas of tissue selectively. The mechanism of this selectivity is not well defined but is
accounted for associations with plasma lipoproteins and delivery to the cell via low-density
lipoprotein receptors [4]. Therefore, the porphyrin-protein system could be a model to study
behaviour of porphyrins in organisms. On the other hand, the selectivity of these sensitizers
towards tumour cells is not always sufficient for PDT to be specific for the cancerous tissue
alone. One possible way to improve the ability of the porphyrins to target tumour cells
specifically is to couple them with another agent which can preferentially interact with the
malignant cells. Some lectins exhibit a preferential agglutination of tumour cells and those
with high affinity for porphyrins can be considered as a potential carriers for porphyrin
sensitizers to tumour tissues. Concanavalin A (Con A) – lectin of the jack bean (Canavalia
ensiformis) was found in high concentration in growing tissues and may have role in the
regulation of plant-cell division [5]. Con A has been already used as carrier for targeted
delivery of chemotherapeutic agents, e.g., conjugates of Con A and α-chain of diphteria toxin
or ricin have been prepared and tested for targeting the toxin to tumour cells [6]. Due to these
properties Con A can be considered as a potential carrier of porphyrin to cancerous tissue in
photodynamic therapy.
The main purpose of our studies included examination of lectin-porphyrin complex
formation using absorption and fluorescence spectroscopy. The present work was concerned
on the two water-soluble cationic porphyrins: tetrakis[4-(trimethylammonio)phenyl]
(H2TTMePP), tetrakis (1-methyl-4-pyridyl) (H2TMePyP), the corresponding Cu(II)
derivatives (CuTTMePP and CuTMePyP) and two water-soluble anionic porphyrins:
tetrakis (4-sulfonatophenyl) (H2TPPS) and tetrakis (4-carboxyphenyl) (H2TCPP).
2. EXPERIMENTAL
Absorption spectra were taken with a SPECORD M42 (Carl Zeiss Jena)
spectrophotometer using quartz cells between 200 and 700 nm at the temperature of 21C.
Spectra were registered digitally under the control of the program M500. Changes of
porphyrins fluorescence upon titration with Con A solution were monitored on a FluoroMax 2
spectrofluorimeter at room temperature using excitation at 600 nm and emission at 430 nm.
Con A shows the typical protein fluorescence due to aromatic amino acids when excited at
280 nm. Titrations were carried out using porphyrin concentration range about 10-6 M with
Con A as a ligand (concentration range about 10-3 M) in the solution of TRIS buffer (0.025
M) in different values of pH.
Typical evolution of the absorption and emission spectra of H2TTMePP and
CuTTMePP solutions occurring upon titration with Con A is shown in the Fig 1. Addition of
Con A results in reduction of the Soret band intensity in the absorption spectra with a
concomitant red shift of the band maximum. The change in absorbance values (ΔA), varied
from 1.35 to 0.55, and the change in wavelength (Δλ) from 413.5 to 430.5 nm for H2TTMePP.
Similar behaviour was observed for CuTTMePP. Upon binding to this lectin the fluorescence
intensities of all used porphyrins were found to increase. It is interesting, that in the presence
of lectin fluorescence of CuTTMePP is observed as opposed to free metalloporhyrin.
A
Absorbance
Absorption Spectra
CuTTMePP
1.0
418
Q Bands x 10
0.8
280 H TTMePP 413
Q Bands x 10
2
546
+ con A
1.4
516
+ con A
1.2
552
580 636
1.0
280
0.8
0.6
430
0.6
424
0.4
0.4
0.2
0.2
0.0
0.0
200
250
300
350
400
450
500
550
600
650
700
200
250
300
350
400
450
500
550
600
650
700
800
850
900
Intensity
Emission Spectra
1.6e+6
4.0e+6
CuTTMePP + con A
1.4e+6
651
H2TTMePP + con A 648 -> 651
3.5e+6
(exc=430 nm)
(exc =430 nm)
1.2e+6
3.0e+6
1.0e+6
2.5e+6
8.0e+5
2.0e+6
6.0e+5
603 -> 609
1.5e+6
611
699 -> 710
714
4.0e+5
1.0e+6
2.0e+5
5.0e+5
0.0
0.0
400
450
500
550
600
650
700
750
800
850
900
400
450
500
550
600
650
700
750
[nm]
Fig. 1. Evolution of the H2TTMePP and CuTTMePP spectra upon titration with Con A
in TRIS solution.
For the calculation of the association constant of the reaction:
with the equilibrium constant (Kn) written as:
ConA
ConA
P  ConA  PConA
 P(ConA) 2  ...
 P(ConA) n
[ P(ConA) n ]
Kn 
[ P(ConA) n 1 ][ConA]
experimental data were fitted using non-linear regression method [7, 8] to the equation:
(1)
A
 0   1 K1 [ConA]   2 K1 K 2 [ConA]2  ...   n K1 K 2 ...K n ...K n [ConA]n
1  K1 [ConA]  K1 K 2 [ConA]2  ...  K1 K 2 ...K n [ConA]n
[ P]
(2)
where A is absorbance; 0 is the molecular absorbance coefficient for the starting porpyrin; 1
and K1, 2 and K2, ... , etc. are molecular absorbance coefficient and association constants for
complexes with stoichiometry 1:1, 1:2, ... , etc., respectively; [ConA] and [P] are analytical
concentration of concanavalin A and porphyrin (with assumption that [ConA] >> [P]).
3. RESULTS AND DISSCUSION
Non-linear least squares fitting, used to calculate K’s from the observed absorbance
changes, was performed for the complexes with various stoichiometry, but only results for 1:1
complexes gave satisfactory accordance between experimental points and calculated curve
(Fig.2). The values of the association constants (K) for the complexes studied are presented in
Table 1, results show that the strength of association varies with pH. The calculated constants
are in good agreement with data known from the literature for the similar pairs of the
interacting porphyrins and peptides [9].
1.2
Absorbance in the Soret band
H2TTMePP + Con A [TRIS]
pH 8.7
pH 2.8
pH 10
1.0
0.8
0.6
0.4
0.2
0
20
40
60
80
100
120
140
160
180
Fig. 2. Plot of the
absorbance
changes at the
Soret band of
H2TTMePP upon
titration with Con
A in solutions of
different
pH;
points are experimental; curves are
generated from the
equation 2.
6
CM Con A * 10
Tab. 1. Stability constants (K) of Con A-porphyrin complexes in different values of pH.
PORPHYRIN
H2TTMePP
H2TMePyP
CuTTMePP
pH
2.80
8.70
10.0
2.80
8.70
10.0
2.80
8.70
K [dcm3·mol-1]
1.00·103 (3%)
4.35·105 (9%)
1.50·106 (8%)
7.41·103 (9%)
6.02·106 (5%)
5.01·106 (7%)
1.48·106 (10%)
6.55· 106 (12%)
CuTMePyP
H2TPPS
H2TCPP
10.0
8.70
8.70
8.70
6.94· 106 (11%)
1.78· 106 (12%)
1.80· 105 (9%)
2.13· 106 (10%)
Interactions between Con A and water-soluble cationic and anionic porphyrins have
been studied spectrosopically. Increase in fluorescence intensity and the red-shift in the
absorption and emission maxima have been observed. As the conclusion of our preliminary
results we can say that porphyrins can associate with concanavalin A, in all cases one
molecule of Con A forms complex with one molecule of porphyrin and the strength of
association increases with increasing pH.
The last observation can be explained by the various degree of porphyrin protonation
in buffer with different pH and by the conformation of Con A also depending on pH. It has
been reported that between pH 4 and 5 Con A exists as a dimer and at pH above 7 it is
predominantly tetrameric. Our results also indicate that concanavalin A, relatively easy to
extract from the plants, can potentially serve as supporting drug-delivery agent for porphyrin
sensitizers in photodynamic therapy.
As an explanation of Cu(II) porphyrins fluorescence we can consider two phenomena:
- decomposition of Cu(II) porphyrin complexes; on the other hand copper complexes
with those porphyrins are extremely strong (they resist even concentrated H2SO4 and
could be easy formed from EDTA solution) and it is hard to believe that protein can
decompose such stable complex,
- the protein acts as additional ligand and in the formed protein-porphyrin 1:1
complexes emission from CuTTMePP and CuTMePyP energy levels are possible.
References:
[1]
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J.P. Reyftmann, P.Morliere, S. Goldstein, R. Santus, L. Dubertret, D. Lagrange,
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(1970), p.93
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