Katarzyna Polska, Stanislaw Radzki

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Formation of the Porphyrin-Protein Complexes in
Water Solution and Sol-Gel Materials

• Katarzyna Polska, Stanislaw Radzki
• Department of Inorganic Chemistry,
• Maria Curie-Sklodowska University
• Pl. M. C. Sklodowskiej 2, 20-031 Lublin, Poland

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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.
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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).
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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
CONCANAVALIN A

a
b
Fig.1. Structure of Concanavalin A protomer (a)
and 11 H2TTMePP-Con A complex (b).
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CATIONIC PORPHYRINS
H2TTMePP H2TMePyP
5,10,15,20-tetrakis 4-trimethyl ammonio)phenyl
porphyrin
5,10,15,20-tetrakis 4-(1-methyl- 4-pyridyl)
porphyrin
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SOL-GEL PREPARATION
Con AH2P

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Con A (CM 110-4)
H2TTMePP Con A 11 (CM 10-4/10-4)
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H2TTMePP (CM 10-4/10-4)
TEOS
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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).
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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.
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1H,1H COSY NMR
H2TTMePP (10-3M)
H2TTMePP Con A (21)

H2TTMePP Con A (11)
H2TTMePP Con A (12)
Con A (10-3M)
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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.
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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.