katarzyna polska , stanisław radzki department of inorganic chemistry,
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Katarzyna Polska , Stanisław Radzki Department of Inorganic Chemistry, Maria Curie-Skłodowska University Pl. M. C. Skłodowskiej 2, 20-031 Lublin, Poland. Formation of the Porphyrin-Protein Complexes in Water Solution and Sol-Gel Materials. PURPOSE. - PowerPoint PPT PresentationTRANSCRIPT
Katarzyna Polska, Stanisław RadzkiDepartment of Inorganic Chemistry, Maria Curie-Skłodowska University Pl. M. C. Skłodowskiej 2, 20-031 Lublin, Poland
Formation of the Porphyrin-Protein Complexes in Water
Solution and Sol-Gel Materials
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.
PURPOSE
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
Fig.1. Structure of Concanavalin A protomer (a)
and 1:1 H2TTMePP-Con A complex (b).
ab
H2TTMePP H2TMePyP
5,10,15,20-tetrakis [4-trimethyl ammonio)phenyl] porphyrin
5,10,15,20-tetrakis [4-(1-methyl-4-pyridyl)] porphyrin
CATIONIC PORPHYRINS
Mixing of TEOS sol& ConA-H2P solution
Gel formation Aging
TEOSsol
ConAH2P
SOL-GEL PREPARATION
Con A–H2P
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
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.
10 9 8 7 6 5 4 3 2 1
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
5.5
6.0
6.5
7.0
7.5
8.0
8.5
9.0
9.5
10.0
11 10 9 8 7 6 5 4 3 2 1 0 -1
0
1
2
3
4
5
6
7
8
9
10
11
11 10 9 8 7 6 5 4 3 2 1 0 -1
0
1
2
3
4
5
6
7
8
9
10
11
11 10 9 8 7 6 5 4 3 2 1 0 -1
0
1
2
3
4
5
6
7
8
9
10
11
H2TTMePP (10-3M)
H2TTMePP + Con A (1:1)
H2TTMePP + Con A (2:1)
H2TTMePP + Con A (1:2)
11 10 9 8 7 6 5 4 3 2 1 0 -1
0
1
2
3
4
5
6
7
8
9
10
11
Con A (10-3M)
1H,1H COSY NMR
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.
CONCLUSIONS
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.