Film formation from colloidal dispersions stabilized by sugar derivatives and their controllable release for selective protein adsorption

Although the use of sugar and sugar derivatives has been documented in polymer research for many years, there are no reports that would utilize these species as polymerization sites of colloidal polymeric particles that, later on, may be released during particle coalescence to form films with surfaces that differentiate protein adsorption. These studies show that, when n-dodecyl-beta-D-maltoside (DDM) is utilized for the synthesis and stabilization of poly[methyl methacrylate-co-(n-butyl acrylate)] (p-MMA/nBA) colloidal particles, upon particle coalescence DDM stratifies near the film-air (F-A) interface. By using attenuated total reflectance Fourier transform infrared (ATR FT-IR) spectroscopy and internal reflection infrared imaging (IRIRI), comparative adsorption studies on p-MMA/nBA surfaces exposed to globulin (Glo), fibrinogen (Fib), and bovine serum albumin (BSA) reveal that the presence of DDM selectively inhibits Glo and Fib adsorption, but does not affect BSA. The presence of DDM also enhances the rate of mobility of sodium dioctylsulfosuccinate (SDOSS) resulting from interactions between DDM and SDOSS moieties, and the surface morphologies change as a result of concentration variations of DDM in the colloidal dispersions.     

Stimuli-responsive surfactant/phospholipid stabilized colloidal dispersions and their film formation

Methyl methacrylate (MMA) and n-butyl acrylate (nBA) were copolymerized into stable colloidal particles in the presence of micelle forming sodium dioctyl sulfosuccinate (SDOSS) and liposome forming 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC) in aqueous media that serve as thermodynamically stable loci for lipophilic monomers and nanostructured templates. These studies show for the first time that hollow colloidal particles may coalesce to form polymeric films and the combination of SDOSS and DLPC dispersing agents provides a stimuli-responsive environment during film formation through which individual surface stabilizing components can be driven to the film-air (F-A) or film-substrate (F-S) interface. Attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) of p-MMA/nBA colloidal dispersions revealed preferential and enhanced mobility of SDOSS and DLPC lipid rafts to the F-A and F-S interfaces in response to thermal, ionic, and enzymatic stimuli.     

Release of phospholipids from colloidal particles to polymeric surfaces

This study presents a small-scale polymerization of high molecular weight methyl methacrylate/n-butyl acrylate (MMA/n-BA) colloidal particles that are synthesized in an aqueous environment in the presence of phospholipid hydrogenated soybean phosphatidylcholine (HSPC) molecules that also serve as the particle stabilizing agents. When such particles coalesce to form polymeric films, they release phospholipids, which, in turn, form organized structures near the film-air (F-A) interface. Diffusion and mobility of phospholipid molecules are affected not only by their compatibility with colloidal particles but also by electrolyte environments of colloidal dispersions. When Na(+), K(+), and Ca(2+) counterions are added to MMA/n-BA aqueous colloidal dispersions stabilized with HSPC, and such films are coalesced, different degrees of diffusion of HSPC to the F-A interface exist, depending on the counterion, and conformational changes of HSPC result. For example, in the presence of Ca(2+), HSPC molecules collapse entropically to form random surface layers, as opposed to smaller Na(+) and K(+), which force amphiphilic HSPC ends to align preferentially parallel to the film surface. These studies show that it is possible to design stimuli-response colloidal systems triggered by chemical environments of active molecules on colloidal polymer particles.     

Immunolabeling efficiency of protein A-gold complexes

A systematic study of the adsorption of protein A on colloidal gold particles varying in size from 5-16 nm was performed at different protein concentrations. The number of protein A molecules bound per colloidal particle was evaluated and the Scatchard analysis of the adsorption parameters was applied for each size of the colloid. The binding of protein A to the colloidal gold surface exhibited the same affinity pattern for all of the particle sizes. At low concentrations of stabilizing protein, adsorption took place with high affinity (Kd 1.96-3.3 nM) and the maximum number of protein A molecules attached with this affinity correlated well with the surface of the particle. At higher concentrations of protein A, adsorption exhibited a significantly lower affinity (Kd 530-800 nM), and no saturation was recorded. Competition by albumin did not reveal a preferential removal of the "low-affinity" bound protein A molecules, contradicting the model of successive shells of stabilizing protein around the colloidal particle. The immunolabeling efficiency of conjugates having the same size of gold nucleus but carrying different numbers of protein A molecules was comparatively investigated by quantitative post-embedding immunocytochemistry. Protein A-gold formed with 5-10-nm colloids gave the highest intensity of labeling when carrying the maximum number of protein A molecules that could be adsorbed with high affinity. Overloading as well as underloading these complexes resulted in a significant decrease of their immunoreactivity. The most efficient conjugates were obtained when stabilization was performed with 6 micrograms protein A/ml gold sol of 5 and 10 nm particle diameter, and 15 micrograms protein/ml of 15-nm colloid.     

Preferential association of glycoproteins to the euchromatin regions of cross-fractured nuclei is revealed by fracture-label

We used fracture-label to establish ultrastructural localization of glycoproteins in cross-fractured nuclei of duodenal columnar and exocrine pancreatic cells. Mannose residues were detected in cell nuclei by labeling freeze-fractured tissues with concanavalin A-horseradish peroxidase X colloidal gold (Con A-HRP X CG) or direct concanavalin A X colloidal gold (Con A X CG); fucose residues were detected with Ulex Europaeus I X colloidal gold (UEA I X CG) markers. Areas of the three main intranuclear compartments (euchromatin, heterochromatin, and nucleolus) exposed by freeze-fracture were determined by automated image analysis. Colloidal gold particles bound to each nuclear subcompartment were counted and the results expressed in number of colloidal gold particles per square micrometer +/- SEM. Duodenal and pancreatic tissues fractured and labeled with Con A-HRP X CG complex or direct Con A X CG conjugates showed that the vast majority of Con A binding sites was confined to euchromatin regions with only sparse labeling of the heterochromatin and nucleolus. UEA I labeling of duodenal columnar cells showed that colloidal gold particles were almost exclusively confined to cross-fractured areas where euchromatin is exposed. Trypsinization of the fractured tissues before labeling with Con A and UEA I abolished 95-100% of the original label. Our results show that, within the nucleoplasm, mannose and fucose are residues of glycoproteins preferentially located within the regions of euchromatin.     

Evaluation of two different Concanavalin A affinity adsorbents for the adsorption of glucose oxidase

Concanavalin A (Con A) was selected as ligand and thus immobilized onto two different supports, namely the polymeric Toyopearl and the inorganic silica, with the protection of its binding sites provided during the coupling procedure. The prepared Con A affinity adsorbents were then employed to evaluate their adsorption behaviour for the enzyme glucose oxidase (GOD). The immobilization kinetics showed that the immobilization of Con A on silica supports was much faster than that on Toyopearl supports, which could highly reduce the possibility of the denaturation of Con A. The optimal adsorption conditions for binding of GOD onto the ligand were determined in terms of the pH value and the ionic strength of the adsorption medium. The adsorption isotherms for binding GOD onto two Con A affinity adsorbents fitted well with the Langmuir equation. The maximum adsorption capacity q(m) of Toyopearl Con A and silica Con A were 7.9 mg/ml and 4.9 mg/ml, with a dissociation constant K(d) of 4.8 x 10(-7)M and 2.6 x 10(-6)M, respectively. Due to the less diffusive resistance, silica Con A showed both higher adsorption and desorption rates for GOD when compared with Toyopearl Con A. The nonspecific adsorption of GOD was less than 8% for both end-capped Toyopearl and silica supports. The dynamic adsorption of GOD for five times repeated processes showed a high stability for both prepared adsorbents. All the results indicate a good suitability of both Con A adsorbents for affinity adsorption of GOD.     

Characterization and quantitation of concanavalin a binding by plasma membrane enriched fractions from soybean root

The binding of concanavalin A (Con A) to soybean root membranes in plasma membrane enriched fractions (recovered from the 34/45% interface of simplified discontinuous sucrose density gradients) was studied using a radiochemical assay employing tritiated (³H)-Con A. The effect of lectin concentration, time, and membrane protein concentration on the specific binding of ³H-Con A by the membranes was evaluated. Kinetic analyses showed that Con A will react with membranes in that fraction in a characteristic and predictable manner. The parameters for an optimal and standard binding assay were established. Maximal binding occurred with Con A concentrations in the range of 8 to 16% of the total membrane protein with incubation times greater than 40 min at 22 C. Approximately 10¹⁵ molecules of ³H-Con A were bound per microgram of membrane protein at saturation. Binding was reversible. Greater than 92% of the total Con A bound at saturation was released by addition of α-methyl mannoside.     

Adsorption of gamma-globulin, a model protein for antibody, on colloidal particles

The effect of surface properties on the adsorption of bovine gamma-globulin, a model protein for antibody, was studied. Polystyrene latex (PS), hydrophilic copolymer lattices of styrene/2-hydroxyethyl methacrylate [P(S/HEMA)], styrene/ methacrylic acid [P(S/MAA)] and methyl methacrylate/ 2-hydroxyethyl methacrylate [P(MMA/HEMA)], and colloidal silica were used. The adsorption isotherms of gamma-globulin on these colloidal particles were measured as a function of pH and ionic strength. The hydrophilic particles showed low affinities for gamma-globulin at alkaline pH, while PS showed high affinities for gamma-globulin over the whole range of pH and ionic strength. The gamma-globulin adsorption on hydrophilic particles was highly reversible with respect to the pH and ionic strength compared with that on PS. These differences indicate that the dominant driving forces of adsorption are related to the hydrophilicity of particles. The adsorption isotherms of all colloidal particles showed the plateau values, and the order of maximum values of plateau adsorption was P(S/MAA) > PS or P(S/HEMA), silica > P(MMA/HEMA). Thus, they were also affected by the charged groups and the hydrophilicity of the surfaces. On the other hand, the plateau values of all colloidal particles were more or less symmetrical with a maximum at around the isoelectric point of gamma-globulin at an ionic strength of 0.01. This behavior is attributed to the important role of the lateral interaction between the adsorbed molecules at low ionic strength.     

Molecular dynamics simulation of the cooperative adsorption of barley lipid transfer protein and cis-isocohumulone at the vacuum-water interface

Molecular dynamic simulations have been carried out on systems containing a mixture of barley lipid transfer protein (LTP) and cis-isocohumulone (a hop derived iso-alpha-acid) in one of its enol forms, in bulk water and at the vacuum-water interface. In solution, the cis-isocohumulone molecules bind to the surface of the LTP molecule. The mechanism of binding appears to be purely hydrophobic in nature via desolvation of the protein surface. Binding of hop acids to the LTP leads to a small change in the 3-D conformation of the protein, but no change in the proportion of secondary structure present in helices, even though there is a significant degree of hop acid binding to the helical regions. At the vacuum-water interface, cis-isocohumulone shows a high surface activity and adsorbs rapidly at the interface. LTP then shows a preference to bind to the preadsorbed hop acid layer at the interface rather than to the bare water-vacuum interface. The free energy of adsorption of LTP at the hop-vacuum-water interface is more favorable than for adsorption at the vacuum-water interface. Our results support the view that hop iso-alpha-acids promote beer foam stability by forming bridges between separate adsorbed protein molecules, thus strengthening the adsorbed protein layer and reducing foam breakdown by lamellar phase drainage. The results also suggest a second mechanism may also occur, whereby the concentration of protein at the interface is increased via enhanced protein adsorption to adsorbed hop acid layers. This too would increase foam stability through its effect on the stabilizing protein layer around the foam bubbles.     

Recognition of glycoprotein peroxidase via Con A-carrying self-assembly layer on gold

We have successfully fabricated a self-assembled layer of concanavalin A (Con A) on a gold surface for recognition of glycoproteins. The type IV Con A is covalently bound to 11-mercaptoundecanoic acid (MUA) on gold with a 2-(5-norbornene-2,3-dicarboximido)-1,1,3,3-tetramethyluronium tetrafluoroborate (TNTU) linkage. The binding interaction between glycoproteins and self-assembled Con A is studied using horseradish peroxidase (HRP) as a model glycoprotein. Voltammetric, electrochemical impedance studies, and photometric activity measurements show the presence of both specific and nonspecific bindings of HRP to the Con A interface. The specific binding is attributed to the Con A-sugar interaction where Con A selectively recognizes the glycosylation sites of HRP. The catalytic current of the HRP-loaded electrode, because of catalytic oxidation of thionine in the presence of hydrogen peroxide (H2O2), is found to be proportional to the HRP concentrations in the incubation solution. A linear correlation coefficient of 0.993 was obtained over a wide HRP concentration range of 12.5 microg/mL to 1 mg/mL. The approach described in this study provides a simple yet selective means to immobilize glycoproteins on a solid support. The specific binding achieved is desirable in biosensor fabrication, glycoprotein separation, recognition, and purification as well as in drug-releasing systems.     

Preparation and characterization of PEGyated Concanavalin A for affinity chromatography with improved stability

In order to improve its stability, immobilized Concanavalin A (Con A) on Toyopearl adsorbents was conjugated with monomethoxy poly(ethylene glycol) succinimidyl propionate (mPEG-SPA) with different molecular weight. A colorimetric method using ninhydrin is proposed to determine the degree of PEGylation; this method has proved to be easy applicable and reproducible. The PEGylation reaction was studied in detail to elucidate how parameters such as molar ratio of mPEG-SPA to Con A and molecular weight of mPEG-SPA affect the degree of PEGylation. The adsorption isotherms of glucose oxidase (GOD) onto native and PEGylated Con A adsorbents showed that the modification did not alter substantially the specificity of the carbohydrate binding ability of Con A. However, the binding capacity for GOD was slightly reduced probably due to the steric hindrance caused by mPEG chains. Adsorption kinetic studies revealed a lower adsorption rate after PEGylation which was attributed to the steric effect. The dynamic adsorption capacity for modified Con A depended very much on the degree of PEGylation and the molecular weight of mPEG derivatives. The adsorption capacity could be highly preserved for Toyopearl Con A modified by mPEG2k (90% of the original adsorption capacity) even with a degree of PEGylation up to 20% (the ratio of primary amino groups of PEGylated immobilized Con A to that of native immobilized Con A). Studies show that the binding capacity of PEGylated Con A was highly preserved under mild process conditions. PEGylated Con A also exhibited obviously higher stability against more stressful conditions such as the exposure to organic solvents and high temperatures. Conjugation of Con A with mPEG2k provided better adsorption performance thus has greater potential for application in affinity separation processes compared with mPEG5k. The fact that PEGylation stabilizes the properties of Con A may greatly expand the range of applications of unstable proteins to bioprocessing (e.g. biocatalysis and downstream separation) as well as other protein applications (e.g. medication, industrial use, etc.).     

Preliminary assessment of removal of pyrogenic lipopolysaccharides with colloidal zirconia adsorbents.

Preliminary evaluation of bare or polymer-coated colloidal monoclinic zirconia of nominal particle size 100 nm indicated that it is an effective adsorbent for pyrogenic lipopolysaccharides (LPS) as measured by chemical and Limulus amebocyte lysate (LAL) assays. Zirconia at 50 micrograms ml-1 adsorbed 99.95% of added E. coli O128 LPS. Residual LPS levels below 0.1 ng ml-1 were easily attained. Colloidal zirconia was able to remove LPS from solution in the presence of bovine albumin (BSA). Some LPS contaminating BSA lacked affinity for zirconia. Preadsorption of phosphate onto bare zirconia blocked LPS adsorption. However, phosphated-oligomeric glycidyl (epoxy) pentaerythritol-coated colloidal zirconia could be derivatized with imidazole-containing ligands to produce an LPS-binding surface. Preliminary results of adsorption of LPS by the coated particles indicated a reduced level of LPS binding compared to bare zirconia, probably because the particles aggregated during the derivatization process, reducing the effective surface available for LPS adsorption.     

Prevention of prostaglandin E2-induced desensitization of rat ovarian cyclic AMP response by concanavalin A

Prolonged exposure (> 6 h) of cultured granulosa cells to Prostaglandin E₂ (PGE₂; 1 μg/ml) led to a near-total loss of the cyclic AMP response to subsequent addition of fresh hormone. Pre-treatment of the cells with concanavalin A (ConA; 2.0 μg/ml) for 1 h blocked the desensitizing action of PGE₂, so that the decline in the response was reduced by 60% with the hormone at high concentration (1.0 μg/ml); a full response was preserved at submaximal concentration of PGE₂ (0.1 – 0.3μg/ml). Other lectins (succinyl Con a, peanut agglutinin and, to a lesser extent, phytohemagglutinin and wheat germ agglutinin) had a stabilizing effect similar to that of Con A. Addition of alpha-methyl-mannoside either with Con A or various times following the addition of Con A to the cells prevented the protective effect of Con A. Concomitant treatment with colchicine or cytochalasin B abolished the ability of Con A to prevent PGE₂-induced desensitization.     

Concanavalin A inhibits oral regeneration in Stentor coeruleus through a mechanism involving binding to the cell surface

Concanavalin A (Con A) has been shown to induce delays in oral regeneration in the ciliate Stentor coeruleus. Associated with the delayed oral regeneration is a shedding of the cell's extracellular pellicle with the loss of some pigment granules. It is shown that the delays in oral regeneration are not the result of the pigment shedding. The delays are localized in the earliest stages of oral regeneration prior to stage 4. The delays caused by Con A are completely reversible by the addition of 2 mg/ml alpha-D-methyl mannoside either at the time of Con A exposure or 5 min later. Con A clearly binds to the cell surface as shown by the binding of FITC-Con A and its reversal by alpha-methyl mannoside. Crosslinking of Con A receptor molecules may be responsible for the effects of Con A since succinyl Con A, which does not crosslink these receptors, has no effect on oral regeneration even at double the Con A concentration. Calcium ions are also implicated in the action of Con A because an excess of extracellular calcium (10 mM) completely eliminates the Con A delays when added simultaneously with Con A. Examination of the minimum extracellular calcium concentration required for this effect showed that 2 mM calcium can reverse most of the delays but that 5 mM is necessary to completely reverse the delays caused by Con A. If the addition of calcium is delayed for various times after Con A addition, the extracellular calcium is progressively less effective in reversing the Con A delays.     

Concanavalin A binding to oligosaccharide chain leads to alterations in properties of band 3

Anion transport activity and thermotropic behavior of Band 3 are found to be altered after binding of concanavalin (Con A) to human erythrocyte ghosts and isolated Band 3. At lower Con A concentration, the rate coefficients of anion transport enhance with increasing Con A concentration, while noticeable changes of the largest calorimetric endotherm of human erythrocyte membranes termed the C transition (Band 3) can not be observed. With 50 micrograms/ml of Con A, the rate coefficient of Con A-modified ghosts increases 34.4% in comparison with that of normal ghosts. Binding of Con A in lower concentration to ghosts bring about increase of fluidity of lipid which maybe contribute to increase anion transport via Band 3. At higher Con A concentration, the C transition tend to lower temperature with increase in Con A concentration, the C transition is shifted from 69.25 degrees C to 66.25 degrees C with 2.5 mg/ml Con A. It is suggested that the Con A-modified Band 3 possess a looser structure than normal one.     

Molecular recognition of concanavalin A on mannoside diacetylene lipid monolayer at the air-water interface.

The interaction of p-10,12-pentacosadiyne-1-n-phenylamide alpha-D-mannopyranoside (MPDA) with protein concanavalin A (Con A) was studied at the air/water interface. The expansion of molecular area of PDA (10,12-pentacosadiynoic acid)/MPDA mixed monolayer after injection of Con A in subphase shows strong interaction between Con A and the monolayer. The maximum expansion of molecular area decreases as the molar ratio of MPDA increases due to the steric hindrance effect. By using enzyme mannosidase to cut-off the mannoside headgroup of MPDA, expansion of molecular area was greatly reduced, indicating that the binding of Con A is specific to the mannoside headgroup. The kinetics of the binding fits to the first order bimolecular reaction model. Fluorescence quenching of fluorescein isothiocyanate labeled Con A after injection into the subphase gives a direct proof of the molecular recognition.     

Corneal glycoconjugates: an ultrastructural lectin--gold study

The oligosaccharide chains of cell surface and extracellular matrix glycoconjugates are essential for the biological properties of these molecules. We have, therefore, investigated carbohydrate residues in the rat cornea using biotinylated lectin--gold probes. Fixed corneas were removed and embedded in Lowicryl HM20 or LR White. Ultrathin sections were incubated in one of the lectins: Triticum vulgare (WGA), Canavalia ensiformis (Con A), Griffonia simplicifolia (GS-1), Limax flavus (LFA) and Allomyrina dichotoma (Allo A), followed by streptavidin--gold, or the sections were incubated in cationic colloidal gold. Semi-quantification of gold labelling was determined for corneal endothelium, Descemet's membrane, stroma and epithelium from electron micrographs. WGA and Con A binding sites were expressed either moderately or strongly through out the cornea, suggesting a preponderance of alpha-mannose and N-acetylglucosamine residues. A particular concentration of these sugars was found in Descemet's membrane. In contrast, GS-1 (specific for alpha-galactose) and Allo A (specific for beta-galactose) labelled all regions weakly. Sialic acid residues, as defined by LFA labelling and the expression of neuraminidase-sensitive cationic colloidal gold binding sites, were sparsely distributed throughout the stroma, Descemet's membrane and endothelium. In contrast, sialoglycoconjugates were found in significant concentrations in the epithelium. Electron microscopy proved useful in providing new information on the cellular and subcellular localization of these lectin binding sites. © Chapman & Hall     

Specific stimulation of heart sarcolemmal Ca2+/Mg2+ ATPase by concanavalin A

The effects of concanavalin A (Con A) on membrane Ca2+/Mg2+ ATPase activities as well as the characteristics of Con A binding were examined by employing rat heart sarcolemmal preparations. Con A stimulated the Ca2+ ATPase and Mg2+ ATPase activities in sarcolemma; maximal stimulation in these parameters was seen at a concentration of 10 micrograms/ml. The observed effects of Con A were blocked by alpha-methylmannoside. Sarcolemmal Na+-K+ ATPase and Ca2+-stimulated ATPase were not affected by Con A. Likewise, Con A did not alter the mitochondrial, sarcoplasmic reticular, and myofibrillar ATPase activities. Con A was found to bind to sarcolemma; alpha-methylmannoside prevented this binding. The Scatchard plot analysis of the data on specific Con A binding showed a straight line with a Kd of about 530 nM and a Bmax of 235 pmol/mg protein, thus indicating that there was only one kind of binding site for Con A in sarcolemma. These results suggest that Con A is a specific activator of the low affinity Ca2+/Mg2+ ATPase system in the heart sarcolemmal membrane.     

Adsorption of RNase A on cationic polyelectrolyte brushes: a study by isothermal titration calorimetry

We present a study of the adsorption of a positively charged protein to a positively charged spherical polyelectrolyte brush (SPB) by isothermal titration calorimetry (ITC). ITC is used to determine the adsorption isotherm as a function of temperature and of salt concentration (at physiological pH 7.2). At low ionic strength, RNase A is strongly adsorbed by the SPB particles despite the fact that both the SPB particles and the protein are positively charged. Virtually no adsorption takes place when the ionic strength is raised through added salt. This is strong evidence for counterion release as the primary driving force for protein adsorption. We calculated that ~2 counterions were released upon RNase A binding. The adsorption of RNase A into like-charged SPB particles is entropy-driven, and protein protonation was not significant. Temperature-dependent measurements showed a disagreement between the enthalpy derived via the van't Hoff equation and the calorimetric enthalpy. Further analysis shows that van't Hoff analysis leads to the correct enthalpy of adsorption. The additional contributions to the measured enthalpy are potentially sourced from unlinked equilibria such as conformational changes that do not contribute to the binding equilibrium.     

Fluoroalcohols induced unfolding of Succinylated Con A: native like beta-structure in partially folded intermediate and alpha-helix in molten globule like state

Concanavalin A (Con A) exists in dimeric state at pH 5. In concentration range 20-60% (v/v) 2,2,2-trifluoroethanol (TFE) and 2-40% (v/v) 1,1,1,3,3,3-hexafluoroisopropanol (HFIP), Con A at pH 5.0 shows visible aggregation. However, when succinyl Con A was used, no aggregation was observed in the entire concentration range of fluoroalcohols (0-90% v/v TFE and HFIP) and resulted in stable alpha-helix formation. Temperature-induced concentration-dependent aggregation in Con A was also found to be prevented/reduced in succinylated form. Possible role of electrostatic repulsion among residues in the prevention of hydrophobically driven aggregation has been discussed. Results indicate that succinylation of a protein resulted in greater stability (in both beta-sheet and alpha-helical forms) against alcohol-induced and temperature-induced concentration-dependent aggregation and this observation may play significant role in amyloid-forming proteins. Effect of TFE and HFIP on the conformation of a dimeric protein, Succinylated Con A, has been investigated by circular dichroism (CD), fluorescence emission spectroscopy, binding of hydrophobic dye ANS (8-anilinonaphthalene-1-sulfonic acid). Far UV-CD, a probe for secondary structure shows loss of native secondary structure in the presence of low concentration of both the alcohols, TFE (10% v/v) and HFIP (4% v/v). Upon addition of higher concentration of these alcohols, Succinylated Con A exhibited transformation from beta-sheet to alpha-helical structure. Intrinsic tryptophan fluorescence studies, ANS binding and near UV-CD experiments indicate the protein is more expanded, have more exposed hydrophobic surfaces and highly disrupted tertiary structure at 60% (v/v) TFE and 30% (v/v) HFIP concentrations. Taken together, these results it might be concluded that TFE and HFIP induce two intermediate states at their low and high concentrations in Succinyl Con A.