Interactions between chondroitin sulfate and concanavalin A

Chondroitin sulfate, the major extracellular matrix glycosaminoglycan, formed an insoluble complex with concanavalin A at pH 5.4 or below. Concanavalin A (500 μg/ml) reacted only with a relatively narrow concentration range of chondroitin sulfate (optimally between 5 and 50 μg/ml) at pH 5.4 in 0.05 M buffer. Similar precipitin-like interactions were seen between concanavalin A and hyaluronic acid or heparin. No precipitating complexes formed between concanavalin A and the glycosaminoglycans at these concentrations in physiological salt solutions (approx. 0.15 M) unless the pH was below 4.5. Precipitating self-aggregates of concanavalin A appeared to be promoted by chondroitin sulfate at pH 7.3, but no significant precipitation occurred between the reactants at this pH even at very high concentrations, nor did soluble complexes form as determined by affinity chromatography on Sephadex G-200 or fractionation on Bio-Gel P-200. Thus, binding between the lectin and glycosaminoglycans appeared to depend upon reversible non-specific electrostatic interactions observed only at low pH and low ionic strength. Stable interactions were not seen in experiments using physiologically balanced salts at near neutral pH.     

Interactions between chondroitin sulfate and concanavalin A.

Chondroitin sulfate, the major extracellular matrix glycosaminoglycan, formed an insoluble complex with concanavalin A at pH 5.4 or below. Concanavalin A (500 microgram/ml) reacted only within a relatively narrow concentration range of chondroitin sulfate (optimally between 5 and 50 microgram/ml) at pH 5.4 in 0.05 M buffer. Similar precipitin-like interactions were seen between concanavalin A and hyaluronic acid or heparin. No precipitating complexes formed between concanavalin A and the glycosaminoglycans at these concentrations in physiological salt solutions (approx. 0.15 M) unless the pH was below 4.5. Precipitating self-aggregates of concanavalin A appeared to be promoted by chondroitin sulfate at pH 7.3, but no significant precipitation occurred between the reactants at this pH even at very high concentrations, nor did soluble complexes form as determined by affinity chromatography on Sephadex G-200 or fractionation on Bio-Gel P-200. Thus, binding between the lectin and glycosaminoglycans appeared to depend upon reversible non-specific electrostatic interactions observed only at low Ph and low ionic strength. Stable interactions were not seen in experiments using physiologically balanced salts at near neutral pH.     

The multiple complexes formed by the interaction of platelet factor 4 with heparin.

The anisotropy of the fluorescence of dansyl (5-dimethylaminonaphthalene-1- sulphonyl) groups covalently attached to human platelet factor 4 was used to detect the macromolecular compounds formed when the factor was mixed with heparin. At low heparin/protein ratios a very-high-molecular-weight compound (1) was formed that dissociated to give a smaller compound (2) when excess heparin was added. 2. A large complex was also detected as a precipitate that formed at high protein concentrations in chloride buffer. It contained 15.7% (w/w) polysaccharide, equivalent to four or five heparin tetrasaccharide units per protein tetramer. In this complex, more than one molecule of protein binds to each heparin molecule of molecular weight greater than about 6 X 10(3).3. The stability of these complexes varied with pH, salt concentration and the chain length of the heparin. The limit complexes found in excess of the larger heparins consisted of only one heparin molecule per protein tetramer, and the failure to observe complexes with four heparin molecules/protein tetramer is discussed.     

Hydrophobicity of lectins. I. The hydrophobic character of concanavalin A.

The hydrophobicity of Concanavalin A has been estimated by its tendency to adsorb to hydrophobic adsorbents. Experiments varying temperature, salt concentration and hydrophobicity of the absorbent were consistent with accepted criteria of hydrophobic interaction between biomolecules and hydrophobic ligands. The biological significance of the hydrophobic character of Concanavalin A is also discussed.     

Physicochemical characteristics of the glycosaminoglycan-lysosomal enzyme interaction in vitro. A model of control of leucocytic lysosomal activity.

1. The activities of 30 different lysosomal enzymes were determined in vitro in the presence of the sulphated glycosaminoglycans, heparin and chondroitin sulphate, all the enzymes being measured on a density-gradient-purified lysosomal fraction. 2. Each enzyme was studied as a function of the pH of the incubation medium. In general the presence of sulphated glycosaminoglycans induced a strong pH-dependent inhibition of lysosomal enzymes at pH values lower than 5.0, with full activity at higher pH values. However, in the particular case of lysozyme and phospholipase A2 the heparin-induced inhibition was maintained in the pH range 4.0-7.0. 3. For certain enzymes, such as acid beta-glycerophosphatase, alpha-galactosidase, acid lipase, lysozyme and phospholipase A2, the pH-dependent behaviour obtained in the presence of heparin was quite different to that obtained with chondroitin sulphate, suggesting the existence of physicochemical characteristic factors playing a role in the intermolecular interaction for each of the sulphated glycosaminoglycans studied. 4. Except in the particular case of peroxidase activity, in all other lysosomal enzymes measured the glycosaminoglycan-enzyme complex formation was a temperature-and time-independent phenomenon. 5. The effects of the ionic strength and pH on this intermolecular interaction reinforce the concept of an electrostatic reversible interaction between anionic groups of the glycosaminoglycans and cationic groups on the enzyme molecule. 6. As leucocytic primary lysosomes have a very acid intragranular pH and large amounts of chondroitin sulphate, we propose that this glycosaminoglycan might act as molecular regulator of leucocytic activity, by inhibiting lysosomal enzymes when the intragranular pH is below the pI of lysosomal enzymes. This fact, plus the intravacuolar pH changes described during the phagocytic process, might explain the unresponsiveness of lysosomal enzymes against each other existing in primary lysosomes as well as its full activation at pH values occurring in secondary lysosomes during the phagocytic process.     

Osmotic second virial cross‐coefficient measurements for binary combination of lysozyme,ovalbumin, and α‐amylase in salt solutions

Interactions measurement is a valuable tool to predict equilibrium phase separation of a desired protein in the presence of unwanted macromolecules. In this study, cross‐interactions were measured as the osmotic second virial cross‐coefficients (B₂₃) for the three binary protein systems involving lysozyme, ovalbumin, and α‐amylase in salt solutions (sodium chloride and ammonium sulfate). They were correlated with solubility for the binary protein mixtures. The cross‐interaction behavior at different salt concentrations was interpreted by either electrostatic or hydrophobic interaction forces. At low salt concentrations, the protein surface charge dominates cross‐interaction behavior as a function of pH. With added ovalbumin, the lysozyme solubility decreased linearly at low salt concentration in sodium chloride and increased at high salt concentration in ammonium sulfate. The B₂₃ value was found to be proportional to the slope of the lysozyme solubility against ovalbumin concentration and the correlation was explained by preferential interaction theory. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1203–1211, 2013     

Immunochemical study of the heterogeneity of trophoblastic beta 1-glycoprotein

It has been found that in mild acid (pH 6.6) and mild-alkaline media (pH 7.7) both pregnancy proteins form complete precipitates. In more alkaline buffer solutions the form of alpha 2-glycoprotein (alpha 2-GP) precipitate is preserved, while trophoblastic beta 1-glycoprotein (TBG) shows three immunochemically identical components with different electrophoretic mobility. The form with beta-globulins mobility predominates, and minor fragments are presented by alpha- and gamma-components. All TBG forms are clearly seen at pH 8.6. In more alkaline medium (pH 10.0) the clarity of the precipitates drastically decreases. It is shown that heparin introduction into the gel of first dimension electrophoresis increases anode electrophoretic mobility of both proteins at polysaccharide concentration of at least 0.1 mg/ml. Large amounts of heparin cause the increase in TBG alpha-component precipitate area and the decrease in the form with beta-globulins mobility. At the same time alpha 2-GP precipitate area and form remain unchanged.     

Protein-membrane interactions: specific vs. non-specific adsorption and binding of proteins and polyamino acids on erythroblasts transformed by Friend virus

Friend-virus transformed erythroblasts (HFL cells) were incubated in solutions containing up to 3 different proteinaceous compounds at pH 7.2 or 5.5 at 5 degrees C. The specificity of interaction of each compound with the cell surface was determined by comparing the amounts of each compound adsorbed and bound in the presence of 2 or 3 different compounds or after prior binding of another compound to the amounts when the individual compounds alone interacted with the cells. At pH 7.2, ovalbumin and gelatin apparently interacted with cell surface components common to both proteins, as indicated by a decrease (up to 80%) in the amount of each compound adsorbed and bound in the presence, or after the binding, of the other compound. The relative amounts of each compound that interacted were different at pH 5.5 and pH 7.2. Gelatin and poly-L-lysine interacted mainly with different components at pH 7.2, whereas common components appeared to be involved at pH 5.5. Concanavalin A interacted preferentially with components that it shared with lysozyme at both pH values. In addition to variations in interactions with changes in pH and type of compound, variations occurred with changes in concentration of the compounds and with their sequence of addition to the cells. Comparative studies with horse red blood cells showed that the interactions differed markedly with cell type.     

Preferential interactions determine protein solubility in three-component solutions: the MgCl2 system

The correlation between protein solubility and the preferential interactions of proteins with solvent components was critically examined with aqueous MgCl2 as the solvent system. Preferential interaction and solubility measurements with three proteins, beta-lactoglobulin, bovine serum albumin, and lysozyme, resulted in similar patterns of interaction. At acid pH (pH 2-3) and lower salt concentrations (less than 2 M), the proteins were preferentially hydrated, while at higher salt concentrations, the interaction was either that of preferential salt binding or low salt exclusion. At pH 4.5-5, all three proteins exhibited either very low preferential hydration or preferential binding of MgCl2. These results were analyzed in terms of the balance between salt binding and salt exclusion attributed to the increase in the surface tension of water by salts, which is invariant with conditions. It was shown that the increase in salt binding at high salt concentration is a reflection of mass action, while its decrease at acid pH is due to the electrostatic repulsion between Mg2+ ions and the high net positive charge on the protein. The preferential interaction pattern was paralleled by the variation of protein solubility with solvent conditions. Calculation of the transfer free energies from water to the salt solutions for proteins in solution and in the precipitate showed dependencies on salt concentration. This indicates that the nature of interactions between proteins and solvent components is the same in solution and in the solid state, which implies no change in protein structure during precipitation. Analysis of the transfer free energies and preferential interaction parameter in terms of the salting-in, salting-out, and weak ion binding contributions has led to the conclusions that, when the weak ion binding contribution is small, the predominant protein-salt interaction must be that of preferential salt exclusion most probably caused by the increase of the surface tension of water by addition of the salt. A necessary consequence of this is salting-out of the protein, if the protein structure is to remain unaltered.     

Interaction of fibrinogen with detergent.

Both cationic and anionic detergents were found to precipitate fibrinogen by forming fibrinogen-detergent complexes. These complexes were soluble in distilled water, but the aqueous solutions were very unstable and the complexes precipitated in the presence of salt. In the interaction of fibrinogen with the cationic detergent, stearyltrimethyl-ammonium chloride, approximately 160 molecules of detergent were found to bind to one molecule of fibrinogen. In distilled water, the fibrinogen-stearyltrimethylammonium complex (FG-STA(Cl)) remained soluble in the presence of thrombin [ED 3.4.21.5] although the same peptides were released as those released from fibrinogen. Precipitation of FG-STA(Cl) by salt was found to be closely related to adsorption of the anion of the salt by the complex. Futher addition of salt resulted in solubilization of the precipitate, and the solubilization was also due to further adsorption of the anion onto the precipitate.     

The interaction of human tryptase-beta with small molecule inhibitors provides new insights into the unusual functional instability and quaternary structure of the protease

Human tryptase-beta (HTbeta) is a serine protease with an atypical tetrameric structure and an unusual dependence on heparin binding or high salt for functional and structural stability. In the absence of heparin and at physiological salt, pH, and temperature, HTbeta rapidly loses activity by a reversible process that we have called spontaneous inactivation. The role of tetramer dissociation in this process is controversial. Using small irreversible or competitive inhibitors of HTbeta as stabilizing ligands, we were able to examine tetramer stability under inactivating (decay) conditions in the absence of heparin and to define further the process of spontaneous inactivation. Size exclusion chromatography showed that interaction with inhibitors stabilized the tetramer. Using sedimentation equilibrium, spontaneously inactivated HTbeta (si-HTbeta) was shown to be a destabilized tetramer that dissociates upon dilution and which in the presence of a competitive inhibitor re-formed a stable tetramer. Addition of inhibitors to si-HTbeta rescued catalytic activity as was shown after inhibitor displacement. At high concentrations of si-HTbeta (4-5 microM), the binding of inhibitor alone provided sufficient free energy for complete reactivation and tetramer stabilization, whereas at low si-HTbeta concentration (0.1 microM) where the destabilized tetramer would be mostly dissociated, reactivation required more free energy which was provided by the binding of both an inhibitor and heparin. The results demonstrate that HTbeta is a tetramer in the absence of heparin and that tetramer dissociation is a consequence of and not a prerequisite for inactivation. Heparin binding likely stabilizes the tetramer by favoring a functionally active conformation with stable intersubunit contacts, rather than by simply cross-linking active monomers.     

Some characteristics of protein precipitation by salts

The solubilities of lysozyme, alpha-chymotrypsin and bovine serum albumin (BSA) were studied in aqueous electrolyte solution as a function of ionic strength, pH, the chemical nature of salt, and initial protein concentration. Compositions were measured for both the supernatant phase and the precipitate phase at 25 degrees C. Salts studied were sodium chloride, sodium sulfate, and sodium phosphate. For lysozyme, protein concentrations in supernatant and precipitate phases are independent of the initial protein concentration; solubility can be represented by the Cohn salting-out equation. Lysozyme has a minimum solubility around pH 10, close to its isoelectric point (pH 10.5). The effectiveness of the three salts studied for precipitation were in the sequence sulfate > phosphate > chloride, consistent with the Hofmeister series. However, for alpha-chymotrypsin and BSA, initial protein concentration affects the apparent equillibrium solubility. For these proteins, experimental results show that the compositions of the precipitate phase are also affected by the initial protein concentration. We define a distribution coefficient kappa(e) to represent the equilibrium ratio of the protein concentration in the supernatant phase to that in the precipitate phase. When the salt concentration is constant, the results show that, for lysozyme, the protein concentrations in both phases are independent of the initial protein concentrations, and thus kappa(e) is a constant. For alpha-chymotrypsin and BSA, their concentrations in both phases are nearly proportional to the initial protein concentrations, and therefore, for each protein, at constant salt concentration, the distribution coefficient kappa(e) is independent of the initial protein concentration. However, for both lysozyme and alpha-chymotrypsin, the distribution coefficient falls with increasing salt concentration. These results indicate that care must be used in the definition of solubility. Solubility is appropriate when the precipitate phase is pure, but when it is not, the distribution coefficient better describes the phase behavior. (c) 1992 John Wiley & Sons, Inc.     

Studies on lectins. XLIV. The pH dependence of lectin interactions with sugars as determined by affinity electrophoresis.

The pH dependence of association constants of the lectin-sugar complexes was determined by means of affinity electrophoresis. All the lectins studied (from the seeds of Dolichos biflorus, Glycine soja, Lens esculenta and Vicia cracca and of the fruiting body of Marasmius oreades) were characterized by a similar course of pH dependence of the association constants, with the maximum values at pH 7--9. For concanavalin A and the L-fucose binding Ulex europaeus lectin only the association constants at three selected pH values were determined. Concanavalin A does not interact with immobilized alpha-D-mannosyl residues at pH 2.3. The association constants vs. pH curves measured for lectins isolated from two different lentil varieties slightly differ in accordance with the differences observed in the interaction of these lectins with the Sephadex gel.     

DNA-binding domains of human plasma fibronectin. pH and calcium ion modulation of fibronectin binding to DNA and heparin

We have studied the binding of fibronectin and its thermolysin fragments to DNA and heparin. Elution of polypeptides bound to DNA-cellulose and heparin-Sepharose affinity chromatography columns was performed by NaCl linear gradients in buffers at different pH and in the presence and absence of calcium ions. The NaCl concentration required to elute fibronectin from both types of column increased as the pH decreased. Fibronectin was not retained on DNA-cellulose or heparin-Sepharose affinity chromatography columns using a buffer containing physiological concentrations of Ca2+, Mg2+ and NaCl, at pH 7.4. On the other hand at pH 6.4 in conditions of physiological ionic strength, fibronectin was retained by both columns, eluting from the DNA-cellulose at 280 mM NaCl and from the heparin-Sepharose column at 210 mM. Furthermore, we have studied the interaction of thermolysin-digested fibronectin both with DNA-cellulose and heparin-Sepharose using the above procedure. The results demonstrate that there are four distinct domains, which interact both with DNA and heparin. We also report here the modulation by pH and Ca2+ ions of the interaction with DNA and heparin of these different domains.     

Competitive inhibition of myosin ATPase activity by different molecular weight heparins.

Myosin ATPase activity was measured, by continuous luminometric method, in presence of different molecular weight heparins. ATPase activity decreases in the presence of heparin, when simultaneous incubation with ATP is carried out; the percentage of inhibition is proportional to polysaccharide concentration. Heparins of different molecular weights (1.75 KD to 11.6 KD) are competitive inhibitors of enzymatic activity; the inhibitory effects is also appreciable with trisulphated disaccharide. The possible mechanisms of interaction between heparin and myosin ATPase are discussed.     

Photoaffinity labeling of concanavalin A. Preparation of a concanavalin A derivative with reduced valence.

Concanavalin A (Con A) was labeled with p-azidophenyl alpha-D-mannopyranoside under ultraviolet irradiation and the reaction products were separated by affinity chromatography on Sephadex G-100 at pH 5. One of the Con A derivatives thus obtained was characterized as a monovalent dimer at pH 5 and a divalent tetramer at pH 7 by sedimentation equilibrium and equilibrium dialysis, indicating that this photoaffinity labeling did not alter the quaternary structure of Con A. In agreement with these results, the labeled Con A did not show the capacity to precipitate glycogen at pH 5, but it formed precipitates with glycogen at pH 7. Although its hemagglutinating activity was found to be weaker than that of the native Con A, the dose-response cure of the labeled Con A in the mitogenic stimulation of human peripheral lymphocytes was almost identical to that of the native con A.     

Characterization of heparin binding of human extracellular superoxide dismutase

The C-terminal domain of human extracellular superoxide dismutase (hEC-SOD) plays a crucial role in the protein's interaction with heparin. Here we investigated this interaction in more detail by comparing the heparin-binding characteristics of two variants of hEC-SOD: the two fusion proteins containing the hEC-SOD C-terminal domain and a synthetic peptide homologous to the C-terminal. The interaction studies were performed using a surface plasmon resonance based technique on a BIAcore system. It should be emphasized that this is a model system. However, the kinetic constants, as measured, are valid in a comparative sense. Comparison of affinities for size-fractionated heparins revealed that octa- or decasaccharides are the smallest heparin fragments that can efficiently interact with the C-terminal domain of hEC-SOD. At physiological salt concentration, and pH 7.4, the hEC-SOD/heparin interaction was found to be of a high-affinity type, with an equilibrium dissociation constant, K(d), of 0.12 microM, which is 700 and 10-20 times lower than the K(d) values for the synthetic peptide and the fusion proteins, respectively. However, when an alpha-helical structure was induced in the synthetic peptide, by addition of 10% trifluoroethanol, the K(d) decreased to 0.64 microM. The differences in the K(d) values were mainly governed by differences in the association rate constants (k(ass)). The hEC-SOD/heparin interaction itself was found to have a fairly high dissociation rate constant (0.1 s(-)(1)), and a very high association rate constant (8 x 10(5) M(-)(1) s(-)(1)), suggesting that the interaction is mainly controlled by the association. These results together with circular dichroism spectra of the synthetic peptide suggest that an alpha-helical structure in the C-terminal is essential for optimal binding to heparin and that other parts of hEC-SOD moderate the affinity. Our data also demonstrate that the tetramerization itself does not substantially increase the affinity.     

Analysis of the Interaction between Heparin and Follistatin and Heparin and Follistatin-Ligand Complexes Using Surface Plasmon Resonance

Heparin and related heparan sulfate interact with a number of cytokines and growth factors, thereby playing an essential role in many physiological and pathophysiological processes by involving both signal transduction and the regulation of the tissue distribution of cytokines/growth factors. Follistatin (FS) is an autocrine protein with a heparin-binding motif that serves to regulate the cell proliferative activity of the paracrine hormone, and member of the TGF-β family, activin A (ActA). Follistatin is currently under investigation as an antagonist of another TGF-β family member, myostatin (Mstn), for the promotion of muscle growth in diseases associated with muscle atrophy. In this study, we employ surface plasmon resonance (SPR) spectroscopy to dissect the binding interactions between the heparin polysaccharide and both free follistatin (FS288) and its complexes (FS288-ActA and FS288-Mstn). FS288 complexes show much higher heparin binding affinity than FS288 alone. SPR solution competition studies using heparin oligosaccharides showed that the binding of FS288 and its complex to heparin is dependent on chain length. Full chain heparin or large oligosaccharides, having 18-20 sugar residues, show the highest binding activity for FS288 and the FS288-ActA complex, whereas smaller heparin molecules could interact with the FS288-Mstn complex. These interactions were also analyzed in normal physiological buffers and at different salt concentrations and pH values. Unbound follistatin was much more sensitive to all salt concentrations of >150 mM. The binding of heparin to the FS288-ActA complex was disrupted at 500 mM salt, whereas it was actually strengthened for the FS288-Mstn complex. At acidic pH values, binding of heparin to FS288 and the FS288-ActA complex was enhanced. While slightly acidic pH values (pH 6.2 and 5.2) enhanced the binding of the FS288-Mstn complex to heparin, at pH 4 heparin binding was inhibited. Overall, these studies demonstrate that binding of a specific ligand to FS288 differentially regulates its affinity and behavior for heparin molecules.     

Interactions of sulfated mucopolysaccharides with lectins. Application to the separation of mucopolysaccharide mixtures.

The interaction of sulfated mucopolysaccharides and lectins has been studied by determining the amount of precipitate formed when mucopolysaccharides are added to a solution of concanavalin A or a partially purified lectin preparation from red kidney bean (Phaseolus vulgaris). The amount of insoluble complex obtained when a given mucopolysaccharide is added to a solution of partially purified red kidney bean preparation is pH dependent. The reaction of concanavalin A and heparin has also been studied by adding increasing amounts of mucopolysaccharide to a fixed amount of lectin. This interaction results in the development of a precipitin-like curve and leads to the isolation of a heparin fraction which has been found to be more reactive with respect to formation of a precipitate than the original heparin preparation. Monosaccharides such as α-methyl-d-mannopyranoside and N-acetyl-d-glucosamine which are known to bind specifically to the lectin, greatly inhibit precipitate formation. The interactions between sulfated mucopolysaccharides and lectins have been used to isolate various sulfated mucopolysaccharides.     

The N-terminal of a heparin-binding sperm membrane mitogen possess lectin-like sequence

Glycosaminoglycans like heparin and heparin sulfate in follicular fluid induce changes in the intracellular environment during the spermatozoal functional maturation. We previously reported the isolation, purification and partial characterization of a heparin binding sperm membrane protein (HBSM). In the present study, the amino acids analysis provided evidence of a single sequence, which suggest the homogeneity of the purified HBSM. Fourteen amino acids--(1)A D T I V A V E L D T Y P N(14)--correspond to the amino terminal sequence of Concanavalin A (Con A) and contain 45.2% carbohydrate by weight. HBSM possess mitogenic property on lymphocytes with comparable magnitude to the well-known mitogen; Con A, inducing 83% radiolabel thymidine incorporation in growing lymphocytes. Unlike Con A, there was no agglutination of cell by HBSM upto 5 ng/ml concentration. Interestingly, we found that heparin and chondroitin sulfate-conjugated HBSM inhibit the proliferative activity. Similar effect was also found with an in-house isolate sulfated glycans; G-I (28% sulfate). In contrast, there was no inhibition by the desulfated form; G-ID. Altogether, our data suggest that the mechanism of cell proliferative pathway may be different for HBSM and Con A.