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Formation of the Porphyrin-Protein
Complexes in Water Solution
and Sol-Gel Materials
Katarzyna Polska, Stanisław Radzki
Department of Inorganic Chemistry,
Maria Curie-Skłodowska University
Pl. M. C. Skłodowskiej 2, 20-031 Lublin, Poland
PURPOSE
Studies of lectin-porphyrin interactions can be important from the point of
view of the influence of lectins on porphyrin-containing biomolecules and the
possible application of these conjugates in photodynamic therapy of cancer (PDT).
PDT has attracted a great deal of attention in recent years as a new cancer
treatment that utilizes porphyrins and metalloporphyrins as sensitizers. Porphyrins
preferentially accumulate in tumour cells, when irradiated by light of appropriate
wavelenght, they go into the excited state and cause irreparable damage of cancer
cells. Concanavalin A, lectin of the jack bean (Canavalia ensiformis), was found in
high concentration in growing tissues and have ability to interact preferentially with
transformed (tumour) cells. Due to these properties this protein can be considered
as a potential carrier for 3rd generation photosensitizers to tumour tissues.
Porphyrins have another potential application, they could be used as the
peptide receptors which work in protic solvents. The goal of selective peptide
complexation in aqueous solution was approached only recently, and still needs
considerable progress until artificial receptors come close to the efficiency of
biological systems.
Interactions of several free base porphyrins and their corresponding
copper(II) complexes with lectin (concanavalin A) have been investigated by
spectroscopic techniques. Experiments have been carried out in water solution and
in monolithic silica gels. Porphyrin-protein systems immobilized in monolithic
silica gels (obtained by polycondensation of tetraethoxysilane using sol-gel
technique) have been also examined by atomic force microscopy (AFM). The
present work was concerned on two water-soluble cationic porphyrins: tetrakis [4(trimethylammonio)phenyl] porphyrin (H2TTMePP), tetrakis (1-methyl-4-pyridyl)
porphyrin (H2TMePyP), their complexes with Cu(II) (CuTTMePP, CuTMePyP)
and two water-soluble anionic porphyrins: tetrakis (4-carboxyphenyl) porphyrin
(H2TCPP) and tetrakis (4-sulfonatophenyl) porphyrin (H2TPPS).
CONCANAVALIN A
is a lectin of the jack bean (Canavalia Ensiformis), its
conformation depends on pH, beetwen pH 4 and 5 it exists as a dimer and at pH
above 7 it is predominantly tetrameric
a
b
Fig.1. Structure of Concanavalin A protomer (a)
and 1:1 H2TTMePP-Con A complex (b).
CATIONIC PORPHYRINS
H2TTMePP
5,10,15,20-tetrakis [4-trimethyl
ammonio)phenyl] porphyrin
H2TMePyP
5,10,15,20-tetrakis [4-(1-methyl4-pyridyl)] porphyrin
SOL-GEL PREPARATION
TEOS
sol
ConA
H2P
Mixing of TEOS sol
& ConA-H2P solution
Con A–H2P
Gel formation
Aging
Con A
(CM = 1·10-4)
H2TTMePP + Con A
1:1 (CM = 10-4/10-4)
H2TTMePP
(CM = 10-4/10-4)
TEOS
Fig.2. H2TTMePP immobilized in monolithic silica gels after 7 days, 1 month
and 6 months of drying (concentration = 7.5 x 10-5 M).
SOLUTION
SOL-GEL
413 nm
Soret Band
1.2
Absorbance
280 nm
H2TTMePP + Con A [TRIS]
Absorbance in 413 nm
Absorbance
1.4
1.0
pH 8.7, log K = 5.64
0.8
1.0
0.6
H2TTMePP + Con A
H2TTMePP
2.5
solution
gel before drying
gel after drying
2.0
646 nm
589 nm
513 nm
0.4
553 nm
0.8
0.2
0
20
40
60
80
100
1.5
120
CM Con A x 106
0.6
430 nm
518 nm
1.0
0.4
0.5
0.2
552 nm
592 nm
649 nm
0.0
0.0
250
300
350
400
450
500
550
600
650
700
350
[nm]
400
450
500
550
600
650
4e+6
H2TTMePP + Con A
4e+6
648 -> 651
(exc430 nm)
3e+6
700
 [nm]
Intensity
Intensity
200
9e+6
H2TTMePP + Con A
H TTMePP + Con A
667 nm
2
exc = 430 nm
8e+6
H TTMePP
2
7e+6
6e+6
3e+6
5e+6
2e+6
603 -> 609
4e+6
2e+6
3e+6
699 -> 710
1e+6
2e+6
5e+5
1e+6
0
0
400
450
500
550
600
650
700
750
800
850
900
[nm]
688 nm
400
450
500
550
600
650
700
750
800
850
900
 [nm]
Fig.3. Absorption and emission spectra of H2TTMePP and H2TTMePP/Con A systems
measured in tris solution (pH 8.7) and in monolithic silica gels.
1H,1H
COSY NMR
H2TTMePP (10-3M)
H2TTMePP + Con A (2:1)
0.5
1.0
0
1.5
1
2.0
2.5
2
3.0
3.5
3
4.0
4
4.5
5.0
H2TTMePP + Con A (1:1)
5
5.5
0
6.0
6
6.5
1
7
7.0
7.5
2
8
8.0
3
8.5
9
9.0
4
10
9.5
10.0
10
9
8
7
6
5
4
3
2
1
5
11
Con A (10-3M)
10
9
8
7
6
5
4
3
2
1
0
-1
11
H2TTMePP + Con A (1:2)
6
7
8
0
0
1
1
2
2
3
3
4
4
5
5
6
6
9
10
11
7
7
8
8
9
9
10
10
11
10
9
8
7
6
5
4
3
2
1
0
-1
11
11
10
9
8
7
6
5
4
3
2
1
0
-1
11
10
9
8
7
6
5
4
3
2
1
0
-1
11
CONCLUSIONS
Both anionic and cationic porphyrins were found to interact with the lectin with
comparable affinity, clearly indicating that the charge on the porphyrin does not play any
role in the binding process and that most likely the interaction is mediated by
hydrophobic forces. Upon binding to concanavalin A an increase in porphyrins
fluorescence intensity and a red-shift in absorption and emission maxima have been
observed.
Each lectin subunit was found to bind one porphyrin molecule. The
association constants estimated from absorption titrations for different porphyrins were
comparable and were in the range 1 x 104 – 7.4 x 106 M-1 at room temperature. The UV-Vis
titrations were carried out in the solution of TRIS buffer with different values of pH (2.8,
8.7 and 10). The strength of association increases with increasing pH and that
observation could be explained by various degree of porphyrin protonation and by the
conformation of concanavalin A, also depending on pH. Concanavalin A is a multimeric
lectin, consisting of non-covalently associated two (below pH 6) or more (above pH 7)
the same subunits.
The sol-gel method allows to manufacture amorphous or crystalline materials
from liquid phase at low temperatures and physiological pHs. Because of the low
temperature growth procedure, dopands, such as fluorescent organic dye molecules, can
be introduced in the solution phase of the sol-gel process to obtain optical materials with
various interesting properties. Biologically important compounds encapsulated in silica
gels have many unique features, as good mechanical durability, high resistance to
chemical and biological degradation and, what is the most important, they retain their
spectroscopic properties and biological activity. The advantages of biologicals captured
in sol-gels might give them applications as biosensors, diagnostic devices and catalysts.