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.