Thermodynamic studies on the interaction of water-soluble porphyrins with the glucose/mannose-specific lectin from garden pea (Pisum sativum)

Due to the application of porphyrins as photosensitizers in photodynamic therapy to treat cancer, and the ability of some lectins to preferentially recognize tumor cells, studies on the interaction of porphyrins with lectins are of considerable interest. Here we report thermodynamic studies on the interaction of several free-base and metallo-porphyrins with pea (Pisum sativum) lectin (PSL). Association constants (Ka) were obtained by absorption titrations by monitoring changes in the Soret band of the porphyrins and the Ka values obtained for various porphyrins at different temperatures are in the range of 1.0 x 10(4) to 8.0 x 10(4) M(-1). Both cationic and anionic porphyrins were found to bind to PSL with comparable affinity. Presence of 0.1 M methyl-alpha-D-mannopyranoside--a carbohydrate ligand that is specifically recognised by PSL--did not affect the binding significantly, suggesting that porphyrin and sugar bind at different sites on the lectin. From the temperature dependence of the Ka values, the thermodynamic parameters, change in enthalpy and change in entropy associated with the binding process were estimated. These values were found to be in the range: delthaH degree = -95.4 to -33.9 kJ x mol(-1) and deltaS degree = -237.2 to -32.2 J x mol(-1) x K(-1), indicating that porphyrin binding to pea lectin is driven largely by enthalpic forces with the entropic contribution being negative. Enthalpy-entropy compensation was observed in the interaction of different porphyrins to PSL, with the exception of meso-tetra-(4-sulfonatophenyl)porphyrinato zinc(II), emphasizing the role of water structure in the overall binding process. Circular dichroism and differential scanning calorimetric studies indicate that while porphyrin binding does not induce significant changes in the lectin structure and thermal stability, carbohydrate binding induces moderate changes in the tertiary structure of the protein and also increases its thermal unfolding temperature and the enthalpy of the unfolding transition.     

Interaction of aromatic imino glycoconjugates with jacalin: experimental and computational docking studies

Altering the lectin properties by chemically modified glycoconjugates can have profound effect on their biological applications. In the present case, jacalin has been chosen to study the binding aspects toward glycoconjugates modified by connecting aromatic moieties through imine conjugation at their C-1- or C-2-positions. Out of 10 glycoconjugates, the galactosyl-naphthyl imine (1c) was found to be most effective toward agglutination inhibition (260 times better than galactose), quenching fluorescence intensity, and exhibiting greater binding (K_a, 1.3 × 10⁴ M⁻¹) with jacalin. The specific binding of galactose conjugates and the nonspecific binding of other conjugates have been demonstrated based on ITC. Changes in the secondary structures have been addressed by far- and near-UV CD spectroscopy. The present studies demonstrated that galactose-based conjugates bind at carbohydrate recognition domain (CRD) mainly through polar interactions in addition to exhibiting some nonpolar/hydrophobic interactions, whereas the conjugates other than galactose primarily interact through hydrophobic interactions. Binding of galactosyl conjugates at CRD has been further demonstrated by rigid docking.     

Thermodynamic analysis of porphyrin binding to Momordica charantia (bitter gourd) lectin.

Owing to the use of porphyrins in photodynamic therapy for the treatment of malignant tumors, and the preferential interaction of lectins with tumor cells, studies on lectin-porphyrin interaction are of significant interest. In this study, the interaction of several free-base and metalloporphyrins with Momordica charantia (bitter gourd) lectin (MCL) was investigated by absorption spectroscopy. Difference absorption spectra revealed that significant changes occur in the Soret band region of the porphyrins on binding to MCL. These changes were monitored to obtain association constants (Ka) and stoichiometry of binding. The tetrameric MCL binds four porphyrin molecules, and the stoichiometry was unaffected by the presence of the specific sugar, lactose. In addition, the agglutination activity of MCL was unaffected by the presence of the porphyrins used in this study, clearly indicating that porphyrin and carbohydrate ligands bind at different sites. Both cationic and anionic porphyrins bind to the lectin with comparable affinity (Ka =10(3)-10(5) m(-1)). The thermodynamic parameters associated with the interaction of several porphyrins, obtained from the temperature dependence of the Ka values, were found to be in the range: DeltaH degrees = -98.1 to -54.4 kJ.mol(-1) and DeltaS degrees =-243.9 to -90.8 J.mol(-1).K(-1). These results indicate that porphyrin binding to MCL is governed by enthalpic forces and that the contribution from binding entropy is negative. Enthalpy-entropy compensation was observed in the interaction of different porphyrins with MCL, underscoring the role of water structure in the overall binding process. Analysis of CD spectra of MCL indicates that this protein contains about 13%alpha-helix, 36%beta-sheet, 21%beta-turn, and the rest unordered structures. Binding of porphyrins does not significantly alter the secondary and tertiary structures of MCL.     

A three-layer ONIOM model for the outside binding of cationic porphyrins and nucleotide pair DNA

In this work we investigated the outside binding mode between a cationic porphyrin and a nucleotide pair of DNA, adenine-thymine and guanine-cytosine, in a supramolecular assembly. We used two structural models (semi-extended, extended) that differ in the size of porphyrin, two kinds of theoretical methods: a three layer ONIOM (B3LYP/6-31G(d)/PM3/UFF), and DFT B3LYP/6-31G(d,p), and three cationic porphyrins. ONIOM method was first tested on the semi-extended model that was calculated in four environments: gas phase, solution phase using an explicit solvent model (H₂O), in the presence of a sodium cation (Na⁺) and in both (H₂O + Na⁺). From interaction energy results, we found that the affinity of the cationic substituent by the adenine nucleotide is favored upon the thymine nucleotide. The extended model that considers the whole porphyrin was applied in the gas phase to the four nucleotides. All the cationic porphyrins showed affinity by the nucleotides in the order adenine > guanine > thymine > cytosine. The interaction energy values for outside binding showed a strong porphyrin-nucleotide interaction (≈-90 kcal?mol^-1), that slightly varies between the nucleotides suggesting that this kind of cationic porphyrin has a little selectivity for some of them. We also found that the effect of the nature of the cationic substituent (chain length) in the porphyrin on the outside binding is small (≈2–13 kcal?mol^-1). Coherence between the results showed that ONIOM is a useful tool to get a reasonable molecular geometry to be used as a starting point in calculations of density functional theory.

Interactions of aromatic mannosyl disulfide derivatives with Concanavalin A: synthesis, thermodynamic and NMR spectroscopy studies

α-d-Mannopyranosyl units were attached to an aromatic scaffold through disulfide linkages to obtain mono- to trivalent glycosylated ligands for lectin binding studies. Isothermal titration calorimetric (ITC) measurements indicated that binding affinities of these derivatives to Concanavalin A (Con A) were comparable to or slightly higher than that of methyl α-d-mannopyranoside (K_a values in the range of 10⁴ M⁻¹). The stoichiometries of the lectin-ligand complexes were in agreement with the formal valencies (1–3) of the respective ligands indicating cross-linking in interactions with the di- and trivalent derivatives. Multivalency effects could not, however, be observed with the latter. These ligands were shown to bind to the carbohydrate binding site of Con A using saturation transfer difference (STD) NMR competition experiments.     

Studies of hematoporphyrin and hematoporphyrin derivative equilibria in heterogeneous systems. Porphyrin-liposome binding and porphyrin aqueous dimerization

Two processes of porphyrins in heterogeneous systems containing aqueous and membrane phases have been studied with hematoporphyrin and hematoporphyrin derivative: Dimerization equilibrium in the aqueous phases and porphyrin-membrane binding equilibrium using liposomes as models for biological membranes. The interrelationship of aqueous aggregations and membrane binding was probed and the porphyrin aggregation state in the membrane, at equilibrium, was assessed. Fluorimetric techniques were employed. The dimerization equilibrium constants, at neutral pH and 37°C were found to be 2.8 · 10⁵ M⁻¹ and 1.9 · 10⁶ M⁻¹ for hematoporphyrin and its derivative, respectively. Over a porphyrin concentration range going from monomer-dominant to dimer-dominant systems, we have found that only monomers are bound to the membrane. The respective monomer-liposome binding constants, found to be independent of the initial monomer/dimer distribution in the aqueous phase, were determined to be 1.6 · 10³ M⁻¹ and 4.1 · 10³ M⁻¹ at neutral pH and 37°C for hematoporphyrin and its derivative, respectively. The monomer-liposome interaction was found to perurb the initial monomer/dimer distribution in the aqueous phase, so that the monomers residing at equilibrium in the membrane originate from both monomers and dimers in the aqueous phase.     

Production of singlet oxygen on irradiation of a photodynamic therapy agent, zinc-coproporphyrin III, with low host toxicity

Zinc-coproporphyrin III (Zincphyrin) acts efficiently as a photodynamic therapy (PDT) agent in mice, while it shows no tumor cell-killing activity in vitro and has a high LD₅₀ (low toxicity) in mice. It appears to have advantages over other porphyrins as a practical PDT reagent. In order to examine the action mechanism of Zincphyrin in PDT, we evaluated the photochemical characteristics of Zincphyrin by measurement of the near-infrared emission at 1268 nm, which provides direct evidence for formation of ¹O₂. Intense emission was observed in the presence of Zincphyrin, and was completely inhibited by NaN₃, a ¹O₂ scavenger. Based on a quenching study, the rate constant of the reaction of ¹O₂ with NaN₃ was determined to be 1.5–3.5 M^–1 s^–1, which is close to the reported value (3.8×10⁸ M^–1 s^–1). The intensity of the ¹O₂-specific emission was proportional to both the laser power and the concentration of Zincphyrin. The fluorescence quantum yield of Zincphyrin was 0.004 in phosphate buffer (100 mM, pH 7.4), which indicates that the excited state decays via other pathway(s) faster than through the fluorescence emission pathway. The lifetime of the triplet state of Zincphyrin (210 s) was relatively long compared to that of other porphyrins, such as hematoporphyrin (Hp) (40 s), coproporphyrin I (50 s), or coproporphyrin III (36 s). These results demonstrate the photodynamic generation of ¹O₂ by Zincphyrin.     

Demonstration of cytoplasmic receptors for the molting hormones in crayfish

Summary From in vitro experiments using different binding assays it is in crayfish demonstrated that the cytosol of target tissues is able to bind both ecdysone and ecdysterone. The ability to bind ecdysteroids is destroyed by heating and by treatment with -chymotrypsin and N-ethyl-maleinimide (NEM) (Figs. 4, 5). In target tissues there is a strong positive correlation between protein content and binding (Fig. 6). The association of the hormone-protein-complex is rapid, taking only a few min even at 5° C (Fig. 3). The binding of the two hormones to the cytosol is both specific and saturable. The association constants for the cytoplasmic receptors from hypodermis, hindgut and midgut gland are in the range of 3–6×10⁷ M^–1 for ecdysone and 5–7×10⁸ M^–1 for ecdysterone (Fig. 8). The data suggest the existence of cytoplasmic ecdysteroid receptors.     

Bacterial adhesion to a model surface with self-generated protection coating of mucin via jacalin

Using a mechanism of `self-generation', polymer surfaces were coated with ocular mucin-type glycoproteins that were extracted from tear fluid and immobilized through specific interaction with a lectin, jacalin. Separately, jacalin affinity chromatography of tear fluid showed the main retained components had molecular weights higher than 200 kDa. In evaluations of bacterial adhesion, a model surface with jacalin-immobilized ocular mucins took up a significantly smaller number of adhered Staphylococcus epidermidis (0.041×10⁶ cells cm^–2) than a bare surface of the same polymer (1.202×10⁶ cells cm^–2). The lectin-mediated ocular mucin coating reduced the bacteria uptake by about 95% showing that the presence of mucin on surfaces may afford a general protection against bacterial colonization.     

Opioid receptor binding in rat spinal cord

The interaction of various radioligands with spinal opioid receptors has been characterized under variable experimental conditions. Binding to , , and sites was measured in all (cervical, thoracic, lumbar) segments. The apparent affinity constant (K) of [³H]Ethylketocyclazocine (EKC) was similar in Tris, 2.09 (±1.06)×10⁸ M^–1, and phosphate buffer, 2.16 (±0.02)×10⁸ M^–1, when its interaction with and sites was blocked. Without blocking ligands, EKC binding was resolved in two components:K ₁=1.01 (±0.21)×10⁹ M^–1 andK ₂=0.95 (±0.61)×10⁷ M^–1. Likewise, the binding of [D-Ala², MePhe⁴, Gly(ol)⁵]enkephalin (DAGO) or [D-Ala², D-Leu⁵]-enkephalin (DADLE) alone was represented by a 2-site model. By adjusting the radioligand and receptor concentration or by the addition of blocking ligands, binding was represented by a 1-site model for DAGO,K=4.35 (±1.41)×10⁸ M^–1, and DADLE,K=2.44 (±0.08)×10⁸ M^–1.The abbreviations used are DADLE [D-Ala², D-Leu⁵]enkephalin - DAGO [D-Ala², MePhe⁴, Gly(ol)⁵]enkephalin - EKC ethylketocyclazocine - DYN dynorphin (1–17)     

Amine uniport at the plasmalemma of charophyte cells II. Ratio of matter to charge transported and permeability of free base

Summary We have previously reported that inward positive current flows across the plasmalemma of voltage-clampedChara cells when ammonia or methylamine is added to the medium. This is attributed to (inward) uniport of the cation. We have measured the stoichiometric ratio of the quantity of methylamine transported to the quantity of positive charge transported. We find 0.9 mol/faraday from pH 5.7 to 8.5, as expected if the cation flux is much larger than that of free base. The ratio increases progressively above pH 9 as the concentration of free base becomes comparable with that of cation: the fluxes fit those predicted if neutral methylamine has a permeability of 1.8 × 10^–5 m/sec. This is comparable with the permeability of the methylammonium ion, 6×10^–6 m/sec, at low concentration and –200 mV, as previously reported. Low concentrations of NH ₄ ⁺ are found to inhibit entry of CH₃NH ₃ ⁺ when membrane PD is constant. Half maximum inhibition is found at 20 m NH ₄ ⁺ , in agreement with the apparentK _M for NH ₄ ⁺ binding to its uniporter site. This suggests that NH ₄ ⁺ and CH₃NH ₃ ⁺ enter by the same uniporter, competing for binding to its binding site.     

α-d-Galactosylation of surface fucoglycoconjugate(s) upon stimulation/activation of murine peritoneal macrophages

Murine resident macrophages express, on their surface, carbohydrate epitopes which undergo changes during their stimulation/activation as monitored by binding of¹²⁵I labelledEvonymus europaea andGriffonia simplicifolia I-B₄ lectins. Treatment of the stimulated macrophages with coffee bean -galactosidase abolished binding of the GS I-B₄ isolectin and changed the binding pattern of theEvonymus lectin. The affinity (K _a) ofEvonymus lectin for -galactosidase-treated macrophages decreased approximately 23-fold, from 1.25×10⁸ M^–1 to 5.5×10⁶ M^–1. Subsequent digestion of -galactosidase-treated macrophages with -l-fucosidase fromTrichomonas foetus, further reduced binding ofEvonymus lectin. Resident macrophages showed the same pattern ofEvonymus lectin binding, with the same affinity, as -galactosidase-treated, stimulated macrophages. These results, together with a consideration of the carbohydrate binding specificity of theEvonymus lectin which, in the absence of -d-galactosyl groups, requires -l-fucosyl groups for binding, indicate the presence, on resident macrophages, of glycoconjugates with terminal -l-fucosyl residues. It is also concluded that during macrophage stimulation/activation -d-galactosyl residues are added to this glycoconjugate and that they form part of the receptor forEvonymus lectin. The same glycoconjugate(s) is/are also expressed on the activated macrophage IC-21 cell line which exhibits the same characteristics as that of stimulated peritoneal macrophages, i.e., it contains -d-galactosyl end groups and is resistant to the action of trypsin. Both lectins were also specifically bound toCorynaebacterium parvum activated macrophages.Abbreviations BSA bovine serum albumin - GS I-B₄ Griffonia simplicifolia I-B₄ isolectin - PBS 0.01m phosphate buffer (pH 7.1) with 0.15m NaCl (unless stated otherwise this buffer contained 3mm azide and was free of divalent cations) - PMSF phenyl methane sulfonyl fluoride - TG thioglycollate brewers medium.     

The Site of Calcium Binding in Relation to the Activation of Myofibrillar Contraction

Skeletal muscle myofibrils, in the presence of 2 mM MgCl₂ at pH 7.0, were found to have two classes of calcium-binding sites with apparent affinity constants of 2.1 x 10⁶ M ^-1 (class 1) and ∼3 x 10⁴ M ^-1 (class 2), respectively. At free calcium concentrations essential for the activation of myofibrillar contraction (∼10^-6 M) there would be significant calcium binding only to the class 1 sites. These sites could bind about 1.3 µmoles of calcium per g protein. Extraction of myosin from the myofibrils did not alter their calcium-binding parameters. Myosin A, under identical experimental conditions, had little affinity for calcium. The class 1 sites are, therefore, presumed to be located in the I filaments. The class 1 sites could only be detected in F actin and myosin B preparations which were contaminated with the tropomyosin-troponin complex. Tropomyosin bound very little calcium. Troponin, which in conjunction with tropomyosin confers calcium sensitivity on actomyosin systems, could bind 22 µmoles of calcium per g protein with an apparent affinity constant of 2.4 x 10⁶ M ^-1. In view of the identical affinity constants of the myofibrils and troponin and the much greater number of calcium-binding sites on troponin it is suggested that calcium activates myofibrillar contraction by binding to the troponin molecule.     

Interaction of Tetrahydrocortisol–Apolipoprotein A-I Complex with Eukaryotic DNA and with Single-Stranded Oligonucleotides

The complex formed by tetrahydrocortisol (THC) and apolipoprotein A-I (ApoAI) specifically interacts with eukaryotic DNA from rat liver. Taken together, physical and chemical data and the results of small-angle X-ray scattering analysis show that interaction of the THC–ApoAI complex with eukaryotic DNA results in deformation of the DNA double helix. Single-stranded fragments were demonstrated to cause deformation of the double helix. In this state DNA forms complexes with DNA-dependent RNA polymerase. This interaction is cooperative and of saturating type; up to six enzyme molecules bind with one DNA molecule. The putative site of complex binding with DNA is the sequence CC(GCC)_n found in many genes including the human ApoAI gene. An oligonucleotide of this type was synthesized. Its association constant (K _a) was 1.66·10⁶ M^–1. Substitution of THC with cortisol considerably decreases the K _a. We suggest that THC interacting with GC pairs of the binding site forms hydrogen bonds with cytosine, inducing rupture of the bonds within the complementary nucleic base pair.     

Concanavalin A interactions with asparagine-linked glycopeptides. The mechanisms of binding of oligomannose,bisected hybrid,and complex type carbohydrates

The affinity of concanavalin A (Con A) for simple saccharides has been known for over 50 years. However, the specificity of binding of Con A with cell-surface related carbohydrates has only recently been examined in detail. Brewer and coworkers [J Biol Chem (1986) 261:7306–10; J Biol Chem (1987) 262:1288–93; J Biol Chem (1987) 262:1294–99] have recently studied the binding interactions of a series of oligomannose and bisected hybrid type glycopeptides and complex type glycopeptides and oligosaccharides with Con A. The relative affinities of the carbohydrates were determined using hemagglutination inhibition measurements, and their modes of binding to the lectin examined by nuclear magnetic relaxation dispersion (NMRD) spectroscopy and quantitative precipitation analyses. The equivalence zones (regions of maximum precipitation) of the precipitin curves of Con A and the carbohydrates indicate that certain oligomannose and bisected hybrid type glycopeptides are bivalent for lectin binding. From the NMRD and precipitation data, two protein binding sites on each glycopeptide have been identified and characterized. Certain bisected complex type oligosaccharides also bind and precipitate Con A, while the corresponding nonbisected analogs bind but do not precipitate the protein. The precipitation data indicate that the bisected complex type oligosaccharides are also bivalent for lectin binding, while the nonbisected analogs are univalent. The NMRD and precipitation data are consistent with different mechanisms of binding of nonbisected and bisected complex type carbohydrates to Con A, including different conformations of the bound saccharides.Abbreviations Con A Concanavalin A with unspecified metal ion content - CMPL Con A with Mn²⁺ and Ca²⁺ at the S1 and S2 sites respectively, in the locked conformation [12]; trisaccharide1, 3,6-di-O-(-d-mannopyranosyl)-d-mannose - -MDM methyl -d-mannopyranoside - NMRD nuclear magnetic relaxation dispersion, the magnetic field dependence of nuclear magnetic relaxation rates, in the present case, the longitudinal relaxation rate, 1/T₁, of solvent protons     

Binding of tetrakis(pyrazoliumyl)porphyrin and its copper(II) and zinc(II) complexes to poly(dG-dC)2 and poly(dA-dT)2

Interactions of cationic porphyrins bearing five-membered rings at the meso position, meso-tetrakis(1,2-dimethylpyrazolium-4-yl)porphyrin (MPzP; M is H₂, Cu^II or Zn^II), with synthetic polynucleotides poly(dG-dC)₂ and poly(dA-dT)₂ have been characterized by viscometric, visible absorption, circular dichroisim and magnetic circular dichroism spectroscopic and melting temperature measurements. Both H₂PzP and CuPzP are intercalated into poly(dG-dC)₂ and are outside-bound to the major groove of poly(dA-dT)₂, while ZnPzP is outside-bound to the minor groove of poly(dA-dT)₂ and surprisingly is intercalated into poly(dG-dC)₂. The binding constants of the porphyrin and poly(dG-dC)₂ and poly(dA-dT)₂ are on the order of 10⁶ M⁻¹ and are comparable to those of other cationic porphyrins so far reported. The process of the binding of the porphyrin to poly(dG-dC)₂ and poly(dA-dT)₂ is exothermic and enthalpically driven for H₂PzP, whereas it is endothermic and entropically driven for CuPzP and ZnPzP. These results have revealed that the kind of the central metal ion of metalloporphyrins influences the characteristics of the binding of the porphyrins to DNA.Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Assessment of the photosensitization properties of cationic porphyrins in interaction with DNA nucleotide pairs

We present a theoretical assessment of the photosensitization properties of meso-mono(N-methylpyridyl) triphenylporphyrin (1, MmPyP⁺), which interacts with DNA nucleotide pairs [adenine (A)-thymine (T); guanine (G)-cytosine (C)] via an external binding mode. The photosensitization properties of the arrangements 1A, 1T, 1G and 1C were investigated. A set of density functionals (B3LYP, PBE0, CAM-B3LYP, M06-2X, B97D) with the 6-31G(d) basis set was used to calculate the electronic absorption spectra in solution (water) following TD-DFT methodology. In all the arrangements, with the exception of 1C, the functional PBE0 produced the lowest deviation of the Soret band (0.1–0.2 eV). Using this functional, we show that the porphyrin–nucleotide interaction is stabilized, as reflected by a larger HOMO–LUMO gap than free porphyrin. A more important effect of the interaction corresponds to the red-shift of the Soret band of MmPyP⁺, which is in agreement with experimental results. This behavior could be explained by the higher symmetry found in arrangements with a lower dipole moment, and by the more symmetrical distribution of electronic density along the molecular orbitals, which provokes electronic transitions of lower energy. The structural model allowed us to show that MmPyP⁺ improves the characteristics as a photosensitizer when it interacts with nucleotide pairs due to the longer wavelength required for the Soret band. Results obtained for porphyrins with larger monocationic substituents (2, MmAP+; 3, MONPP+) do not lead to the same behavior. Although the structural model is insufficient to describe porphyrin photosensitization, it suggests that improvements in this property are produced by the inclusion of a cationic charge in the pyridyl ring and a smaller size of the substituent leading to a better communication in the porphyrin–nucleotide pair.     

Kinetic measurements of binding of galectin 3 to a laminin substratum

Galectin 3, a -galactoside binding protein, contains a C-terminal carbohydrate recognition domain (CRD) and an N-terminal segment including multiple repeats of a proline/tyrosine/glycine-rich motif. Previous work has shown that galectin 3 but not the isolated CRD binds to laminin, a multivalent ligand, with positive cooperativity indicating the formation of multiple interactions although the lectin in solution is monomeric. Using surface plasmon resonance, we find that hamster galectin 3 at sub-µmolar concentrations or its isolated CRD at all concentrations binds to a laminin substratum with similar association (k_ass; 10 – 30 000 M^–1 S^–1) and dissociation (k_diss; 0.2 – 0.3 S ₁ ^–1 ) rates and weak affinity (Ka; 1 - 3 X 10⁵ M^–1). At higher concentrations of galectin 3 the off rate decreases ten fold leading to increased affinity. Ligation of an N-terminal epitope of galectin 3 with a monoclonal Fab fragment increases association and dissociation rates ten fold. A recombinant protein obtained by deletion of the first 93 N-terminal residues binds to laminin with positive cooperativity and a slowly dissociating fraction (K_diss; 0.002 S^–1) accummulates on the substratum. The data suggest that homophilic interactions between CRD as well as N terminal domains are implicated in galectin 3 aggregation on the substratum leading to positive binding cooperativity.     

Binding of concanavalin A to intact cells and spheroplasts ofAnacystis nidulans

Native cells of the cyanobacterium (blue-green alga)Anacystis nidulans did not bind fluorescein isothiocyanate-conjugated concanavalin A (FITC-ConA) as measured by fluorescent spectrophotometry. By contrast, spheroplasts ofA. nidulans underwent rapid and specific agglutination in the presence of ConA thus showing appreciable affinity towards the lectin. After treatment with 0.01–0.05% (wt/vol) cetyltrimethylammonium bromide (CTAB) intact cells also became liable to ConA binding, which was not accompanied by significant agglutination. Detergents, other than CTAB, were far less effective. Specific and nonspecific binding was discriminated, as usual, with the aid of methyl -d-mannoside. Conditions are described that allow specific binding of up to 7×10⁴ molecules of FITC-ConA per cell. The binding of ConA to pretreated cells ofA. nidulans was verified by freeze-etching electron microscopy using ferritin-ConA conjugate. Our results appear to be first to demonstrate lectin binding to a cyanobacterium.     

Thermodynamic and kinetic analysis of porphyrin binding to Trichosanthes cucumerina seed lectin.

The interaction of several metallo-porphyrins with the galactose-specific lectin from Trichosanthes cucumeirna (TCSL) has been investigated. Difference absorption spectroscopy revealed that significant changes occur in the Soret band region of the porphyrins upon binding to TCSL and these changes have been monitored to obtain association constants (Ka) and stoichiometry of binding (n). The dimeric lectin binds two porphyrin molecules and the presence of the specific saccharide lactose did not affect porphyrin binding significantly, indicating that the sugar and the porphyrin bind at different sites. The Ka values obtained for the binding of different porphyrins with TCSL at 25 degrees C were in the range of 2 x 10(3)-5 x 10(5) m(-1). Association constants for meso-tetra(4-sulphonatophenyl)porphyrinato copper(II) (CuTPPS), a porphyrin bearing four negative charges and meso-tetra(4-methylpyridinium)porphyrinato copper(II) (CuTMPyP), a porphyrin with four positive charges, were determined at several temperatures; from the temperature dependence of the association constants, the thermodynamic parameters change in enthalpy (DeltaH degrees ) and change in entropy (DeltaS degrees ) associated with the binding process were estimated. The thermodynamic data indicate that porphyrin binding to TCSL is driven largely by a favourable entropic contribution; the enthalpic contribution is very small, suggesting that the binding process is governed primarily by hydrophobic forces. Stopped-flow spectroscopic measurements show that binding of CuTMPyP to TCSL takes place by a single-step process and at 20 degrees C, the association and dissociation rate constants were 1.89 x 10(4) m(-1).s(-1) and 0.29 s(-1), respectively.