ANS Molecules Research Articles

Thermodynamic study of the dimerization of 8-anilino-1-naphthalenesulfonic acid by isothermal titration calorimetry

Dimerization of the fluorescence probe, 8-anilino-1-naphthalenesulfonic acid (ANS) in aqueous media have been studied by isothermal titration calorimetry (ITC). ITC experiments carried out at different pHs show that dimerization constants are highly pH dependent, decreasing their values with increasing pH. No dimerization is detected over pH 7. Analyzing the dependence of K dim on pH, using a model that only considers dimers between zwitterionic molecules of ANS, a value of 5.6 for the pK of anilinium moiety is obtained. It is in agreement with the pK determined spectrophotometrically. The dimerization process is enthalpically disfavored and entropically driven at all pH and temperatures studied, indicating that hydrophobic effect has an important role on the formation of dimers. Although dimerization constants are low, dimerization equilibria must be taken into account when the energetics of the interaction of ANS to a protein is studied at pH below 7.

Kinetics and Thermodynamics of the Interaction of ANS with Proteins

1-anilino-naphtalene-8-sulfonate (ANS) is a fluorescent probe widely used in protein folding and conformational transitions studies. The fluorescent features of ANS, a blue shift of the emission maximum and the increase of quantum yield and lifetime, are generally attributed to the binding at hydrophobic sites. Despite the interaction of ANS with proteins has been extensively studied since the early works of Gregorio Weber, few high-resolution structures of proteins complexed with ANS have been resolved. In this work the binding of ANS to BSA was analyzed at equilibrium and pre-equilibrium conditions. The combined analysis of fluorescence, near UV circular dichroism and isothermal titration calorimetric data provided a detailed description of the binding mechanism. Three ANS molecules bound to BSA in 100 mM phosphate pH 7 at 25°C. Pre-equilibrium experiments allowed to determine the affinity and the relative quantum yield at each binding site by fitting a microscopic model to the fluorescence time-course data. This analysis unambiguously indicated that the binding of ANS to BSA occurs at two different and independent binding sites with similar quantum yields and affinities (ΔG° @ -35 kJ/mol). The binding of ANS to the first site is thermodynamically favored by similar contributions of the enthalpic (ΔH = -16.3 kJ/mol) and entropic terms (-TΔS = -19.4 kJ/mol), while the binding to the second site is enthalpically driven (ΔH = -36.6 kJ/mol; -TΔS = 4.3 kJ/mol). Complementary information from molecular docking showed 3 ANS molecules bound at hydrophobic cavities in BSA subdomains IIA and IIIA with binding affinities in the order of those found experimentally. The sulfonate group of ANS was oriented towards clusters of polar residues, a common feature in the reported crystal structures of other ANS-protein complexes. Supported with grants from UBA, CONICET and ANPCyT.

Self-assembly of Bacteriophage-associated Hyaluronate Lyase (HYLP2) into an Enzymatically Active Fibrillar Film

The in vitro assembly of a soluble protein into its mature fibrillar form is usually accompanied by loss of its functional activity. Our study is the first demonstration of a natural enzyme (HylP2) retaining its enzymatic activity on conversion from pre-fibril to mature fibril and supports the contention that minor conformational changes in the native folded form of a protein can lead to the formation of a functional fibril. Hyaluronate lyase (HylP2) is a natural enzyme of bacteriophage 10403 of Streptococcus pyogenes. At pH 5.0, the enzyme undergoes partial unfolding localized in its N-terminal domain while the C-terminal domain maintains its folded trimeric conformation. This structural variant of HylP2 retains about 70% enzymatic activity with hyaluronan. It further self-assembles into a fibrillar film in vitro through solvent-exposed nonpolar surfaces and intermolecular beta-sheet formation by the beta-strands in the protein. Interestingly, the mature fibrillar film of HylP2 also retains about 60 and 20% enzymatic activity for hyaluronic acid and chondroitin sulfate, respectively. The possession of broad substrate specificity by the fibrillar form of HylP2 indicates that fluctuations in pH, which do not lead to loss of functionality of HylP2, might assist in bacterial pathogenesis. The formation of fibrillar film-like structure has been observed for the first time among the hyaluronidase enzymes. After acquiring this film-like structure in bacteriophage, HylP2 still retains its enzymatic activity, which establishes that these fibrils are a genuinely acquired protein fold/structure.

Characterization of the binding of 8-anilinonaphthalene sulfonate to rat class Mu GST M1-1

Molecular docking and ANS-displacement experiments indicated that 8-anilinonaphthalene sulfonate (ANS) binds the hydrophobic site (H-site) in the active site of dimeric class Mu rGST M1-1. The naphthalene moiety provides most of the van der Waals contacts at the ANS-binding interface while the anilino group is able to sample different rotamers. The energetics of ANS binding were studied by isothermal titration calorimetry (ITC) over the temperature range of 5–30 °C. Binding is both enthalpically and entropically driven and displays a stoichiometry of one ANS molecule per subunit (or H-site). ANS binding is linked to the uptake of 0.5 protons at pH 6.5. Enthalpy of binding depends linearly upon temperature yielding a Δ C p of − 80 ± 4 cal K − 1 mol − 1 indicating the burial of solvent-exposed nonpolar surface area upon ANS-protein complex formation. While ion-pair interactions between the sulfonate moiety of ANS and protein cationic groups may be significant for other ANS-binding proteins, the binding of ANS to rGST M1-1 is primarily hydrophobic in origin. The binding properties are compared with those of other GSTs and ANS-binding proteins.

The photosensitive properties of polysiloxane acrylate resin containing tertiary amine groups

AbstractThe photosensitive properties of a novel oligomer, di (N,N‐diacrylolyl)‐α, ω‐diaminopolysiloxane (ANS) with tertiary amine groups and acryloyl groups in its molecular structure were investigated using FTIR and gel yield method. It was noted that the ANS system showed a notable photosensitive property and its photosensitivity in air could be up to16.3 mJ/cm2. The UV‐curing behavior of the ANS was studied by electron spin resonance (ESR). The results showed that amino‐alkyl radicals can be formed by excited BP abstracting hydrogen at a‐carbon bonded with nitrogen in the ANS molecule under UV irradiation, which can mitigate the oxygen inhibition in radical polymerization. It is proven that tertiary amine groups introduced into ANS could boost photosensitivity of the photopolymerization system. The oligomer ANS may find application in photopolymerization to improve the properties of UV‐curing coating materials. © 2007 Wiley Periodicals, Inc. J Appl Polym Sci, 2008

Molecular-Recognition-Induced Phase Transitions of Two Thermo-Responsive Polymers with Pendentβ-Cyclodextrin Groups

PNIPAM-based thermo-responsive polymers with pendent β-cyclodextrin groups were synthesized and the molecular-recognition-induced phase-transition behavior of fabricated polymers was investigated. The results showed that the thermo-sensitive PNG-ECD and PNG-HCD polymers could significantly recognize the guest ANS molecules, and their LCSTs in ANS aqueous solutions were lower than those in blank aqueous solution. The more ANS could be recognized by the polymer, the lower was the LCST of the polymer. The guest NS molecules had an opposite influence on the LCSTs of the PNG-ECD and PNG-HCD polymers, because the complexation between CD and NS slightly enlarged the hydrophilic moiety of the polymers.

Evidence for internal and external binding sites on human tear lipocalin

8-Anilino-1-naphthalenesulfonic acid (ANS) is widely used as a probe for locating binding sites of proteins. To characterize the binding sites of tear lipocalin (TL), we studied ANS binding to apoTL by steady-state and time-resolved fluorescence. Deconvolution of ANS binding revealed that two lifetime components, 16.99ns and 2.76ns at pH 7.3, have dissociation constants of 0.58μM and 5.7μM, respectively. At pH 3.0, the lifetime components show decreased affinities with dissociation constants of 2.42μM and ∼21μM, respectively. Selective displacement of ANS molecules from the ANS–apoTL complex by stearic acid discriminates the internal and external binding sites. Dependence of the binding affinity on ionic strength under various conditions provides strong evidence that an electrostatic interaction is involved. Time-resolved fluorescence is a promising tool to segregate multiple binding sites of proteins.

PH‐dependent aggregation of cutinase is efficiently suppressed by 1,8‐ANS

We have studied the thermal stability of the triglyceride-hydrolyzing enzyme cutinase from F. solani pisi at pH values straddling the pI (pH 8.0). At the pI, increasing the protein concentration from 5 to 80 microM decreases the apparent melting temperature by 19 degrees C. This effect vanishes at pH values more than one unit away from pI. In contrast to additives such as detergents and osmolytes, the hydrophobic fluorophore 1,8-ANS completely and saturably suppresses this effect, restoring 70% of enzymatic activity upon cooling. ANS binds strongly to native cutinase as a noncompetitive inhibitor with up to 5 ANS per cutinase molecule. Only the first ANS molecule stabilizes cutinase; however, the last 4 ANS molecules decrease Tm by up to 7 degrees C. Similar pI-dependent aggregation and suppression by ANS is observed for T. lanuginosus lipase, but not for lysozyme or porcine alpha-amylase, suggesting that this behavior is most prevalent for proteins with affinity for hydrophobic substrates and consequent exposure of hydrophobic patches. Aggregation may be promoted by a fluctuating ensemble of native-like states associating via intermolecular beta-sheet rich structures unless blocked by ANS. Our data highlight the chaperone activity of small molecules with affinity for hydrophobic surfaces and their potential application as stabilizers at appropriate stoichiometries.

Elucidating the binding thermodynamics of 8‐anilino‐1‐naphthalene sulfonic acid with the A‐state of α‐lactalbumin: An isothermal titration calorimetric investigation

Isothermal titration calorimetry has been used to demonstrate that the heat profile associated with the binding of 8-anilino-1-naphthalene sulfonic acid (ANS) with the acid induced molten globule state (A-state) of alpha-lactalbumin (alpha-LA) is different from that with the native and denatured states of the protein. The results corroborate the spectroscopic observations that ANS binds more strongly to the partially folded states of the protein compared to that with the native and denatured states. ANS binds to the A-state of alpha-LA at two independent binding sites that remain nearly the same in the temperature range of 10-35 degrees C. The number of moles of ANS binding at site 1 at 10 degrees C is 14.0+/-0.2 and remains nearly the same with rise in temperature. However, the number of moles of ANS molecules binding at site 2 show an increase from 1.6+/-0.2 at 10 degrees C to 4.1+/-0.1 at 35 degrees C. The deviation of the slope of enthalpy-entropy compensation plot from unity and nonadherence to van't Hoff dictates implies that the binding sites on the A-state of alpha-LA for ANS are not well defined and specific; rather, these binding sites are formed due to greater exposure of hydrophobic clusters in the A-state of the protein. The results for the first time demonstrate the use of isothermal titration calorimetry in characterizing the A-state of alpha-LA both qualitatively and quantitatively.

Combined effects of magnetic fields and temperature changes on 1-aminonaphthalene-8-sulfonic acid fluorescence in red blood cell ghost cell membrane

In the present study, we performed an experiment to clarify the possible effects of magnetic fields of up to 8 T on cell membrane fluidity by using red blood cell ghosts and a fluorescence dye, 1-aminonaphthalene-8-sulfonic acid (ANS). The time course of ANS emission at 480 nm under the influence of a magnetic field at 5 T was observed. The effects of multiple rapid temperature changes and magnetic fields were investigated. The emission intensity at 480 nm increased when the temperature of the cell holder was increased from 20 to 38–46 °C for 15 min. A change in temperature exhibited an increase in the fluidity of the lipid molecules in the cell membrane and increased the population of ANS molecules emitting light at 480 nm in the cell membrane, which is hydrophobic. A discontinuous change in fluorescence at 38–40 °C was exhibited under exposure to a magnetic field at 5 T, while the temperature dependency was continuous without exposure to the magnetic field. In addition, under exposure to the magnetic field, the fluorescence during a decrease in temperature from 38 to 20 °C remained at a level close to the fluorescence during an increase in temperature. The results indicated that the fluidity of the molecules in the cell membrane was decelerated by exposure to magnetic fields at 5 T. We speculated that the magnetic orientation in a part of the lipid membrane disturbed the release of ANS molecules from a hydrophobic region of the membrane.

Periplasmic Protein HdeA Exhibits Chaperone-like Activity Exclusively within Stomach pH Range by Transforming into Disordered Conformation

The extremely acidic environment of the mammalian stomach, with a pH range usually between 1 and 3, represents a stressful challenge for enteric pathogenic bacteria such as Escherichia coli before they enter into the intestine. The hdeA gene of E. coli was found to be acid inducible and was revealed by genetic studies to be important for the acid survival of the strain. This study was performed in an attempt to characterize the mechanism of the activity of the HdeA protein. Our data provided in this report strongly suggest that HdeA employs a novel strategy to modulate its chaperone activity: it possesses an ordered conformation that is unable to bind denatured substrate proteins under normal physiological conditions (i.e. at neutral pH) and transforms into a globally disordered conformation that is able to bind substrate proteins under stress conditions (i.e. at a pH below 3). Furthermore, our data indicate that HdeA exposes hydrophobic surfaces that appear to be involved in the binding of denatured substrate proteins at extremely low pH values. In light of our observations, models are proposed to explain the action of HdeA in both a physiological and a molecular context.

Molecular structure of agarose chains in thermoreversible hydrogels revealed by means of a fluorescent probe technique

AbstractThermoreversible hydrogels of agarose with both high and low molecular weights were studied by means of a fluorescence probe method using 1‐anilino‐8‐naphthalene sulfonic acid (ANS). The fluorescence spectra of ANS in agarose/water changed markedly with agarose concentration. Analysis of the spectra shows that this change is not due to a shift of the whole spectrum, but that there are two fluorescent components of ANS in addition to the fluorescence peak at 542 nm due to free ANS molecules in water. The fluorescence component at 461 nm is assumed to be from ANS intercalated within either a single 31 helix‐form agarose chain or nonpolar region produced by chemical defect, and the fluorescence component between 507 and 522 nm is assumed to be from ANS in aggregated regions of agarose chains having a loose helical structure. There is no doubt that a certain number of water molecules are included in the aggregated region. © 2005 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 43: 680–688, 2005

The structural events associated with the binding of divalent cations to β-bungarotoxin

In order to address the mechanism why the Ca 2+ was crucial for the manifestation of the phospholipase A 2 (PLA 2) activity of β-bungarotoxin (β-BuTx), four divalent cations were used to assess their influences on the catalytic activity and the fine structures of β-BuTx. Substitution Mg 2+ or Sr 2+ for Ca 2+ in the substrate solution was found to cause a decrease in the PLA 2 activity to approximately 15 or 6% of that in the presence of Ca 2+. However, only marginally detectable PLA 2 activity was observed with the addition of Ba 2+. The nonpolarity of 8-anilinonaphthalene-1-sulfonate (ANS)-binding site of β-BuTx markedly increased with the binding of cations to β-BuTx. The negative ellipticity noted with the CD spectra of β-BuTx increased upon the binding of cations too. With the exception of Ba 2+, the order of the ability of cations to enhance the intensity of ANS fluorescence or increase the increment of negative ellipticity was Sr 2+>Ca 2+>Mg 2+, which was the same order as the increase in their atomic radii. However, the energy transfer from Trp fluorescence emission to ANS was most effective upon the addition of Ca 2+. Moreover, the extent of glutaraldehyde crosslinking between A chain and B chain decreased in the presence of cations. Nevertheless, the binding affinities of β-BuTx for the four cations were similar. These results, together with the findings that the ANS molecule binds at the active site of the A chain in β-BuTx, suggest that the binding of Ca 2+ to β-BuTx induces subtly conformational changes occurred at the active site for exerting the activity of β-BuTx. Moreover, the change in the gross conformation induced by the binding of Ca 2+ may affect the interaction between A chain and B chain, and consequently the activity of β-BuTx as well.

Perturbation of Protein Tertiary Structure in Frozen Solutions Revealed by 1-Anilino-8-Naphthalene Sulfonate Fluorescence

Although freeze-induced perturbations of the protein native fold are common, the underlying mechanism is poorly understood owing to the difficulty of monitoring their structure in ice. In this report we propose that binding of the fluorescence probe 1-anilino-8-naphthalene sulfonate (ANS) to proteins in ice can provide a useful monitor of ice-induced strains on the native fold. Experiments conducted with copper-free azurin from Pseudomonas aeruginosa, as a model protein system, demonstrate that in frozen solutions the fluorescence of ANS is enhanced several fold and becomes blue shifted relative free ANS. From the enhancement factor it is estimated that, at −13°C, on average at least 1.6 ANS molecules become immobilized within hydrophobic sites of apo-azurin, sites that are destroyed when the structure is largely unfolded by guanidinium hydrochloride. The extent of ANS binding is influenced by temperature of ice as well as by conditions that affect the stability of the globular structure. Lowering the temperature from −4°C to −18°C leads to an apparent increase in the number of binding sites, an indication that low temperature and /or a reduced amount of liquid water augment the strain on the protein tertiary structure. It is significant that ANS binding is practically abolished when the native fold is stabilized upon formation of the Cd 2+ complex or on addition of glycerol to the solution but is further enhanced in the presence of NaSCN, a known destabilizing agent. The results of the present study suggest that the ANS binding method may find practical utility in testing the effectiveness of various additives employed in protein formulations as well as to devise safer freeze-drying protocols of pharmaceutical proteins.

ANS fluorescence as a tool to monitor cross-linking polymerization of acrylamide

A method based on monitoring the fluorescence intensity of 1-anilinonaphthalene-8-sulphonate (ANS) has been introduced to follow the cross-linking polymerization of acrylamide initiated by ammonium persulfate-TEMED redox initiator system. There is about a 20-fold increase in the fluorescence intensity of ANS accompanying polymerization. Preferential location of ANS molecules near the hydrophobic regions of the polymer surface appears to explain the fluorescence intensity enhancement. The method is found to be consistent with the conventional dilatometric method.

Thermodynamics of the ligandin function of human class Alpha glutathione transferase A1-1: energetics of organic anion ligand binding

In addition to their catalytic functions, cytosolic glutathioneS-transferases (GSTs) are a major reserve of high-capacity binding proteins for a large variety of physiological and exogenous non-substrate compounds. This ligandin function has implicated GSTs in numerous ligand-uptake, -transport and -storage processes. The binding of non-substrate ligands to GSTs can inhibit catalysis. In the present study, the energetics of the binding of the non-substrate ligand 8-anilino-1-naphthalene sulphonate (ANS) to wild-type human class Alpha GST with two type-1 subunits (hGSTA1-1) and its ΔPhe-222 deletion mutant were studied by isothermal titration calorimetry. The stoichiometry of binding to both proteins is one ANS molecule per GST subunit with a greater affinity for the wild-type (Kd=65μM) than for the ΔPhe-222 mutant (Kd=105μM). ANS binding to the wild-type protein is enthalpically driven and it is characterized by a large negative heat-capacity change, ΔCp. The negative ΔCp value for ANS binding indicates a specific interface with a significant hydrophobic component in the protein—ligand complex. The negatively charged sulphonate group of the anionic ligand is apparently not a major determinant of its binding. Phe-222 contributes to the binding affinity for ANS and the hydrophobicity of the binding site.

Thermodynamics of the ligandin function of human class Alpha glutathione transferase A1-1: energetics of organic anion ligand binding.

In addition to their catalytic functions, cytosolic glutathioneS-transferases (GSTs) are a major reserve of high-capacity binding proteins for a large variety of physiological and exogenous non-substrate compounds. This ligandin function has implicated GSTs in numerous ligand-uptake, -transport and -storage processes. The binding of non-substrate ligands to GSTs can inhibit catalysis. In the present study, the energetics of the binding of the non-substrate ligand 8-anilino-1-naphthalene sulphonate (ANS) to wild-type human class Alpha GST with two type-1 subunits (hGSTA1-1) and its DeltaPhe-222 deletion mutant were studied by isothermal titration calorimetry. The stoichiometry of binding to both proteins is one ANS molecule per GST subunit with a greater affinity for the wild-type (K(d)=65 microM) than for the DeltaPhe-222 mutant (K(d)=105 microM). ANS binding to the wild-type protein is enthalpically driven and it is characterized by a large negative heat-capacity change, DeltaC(p). The negative DeltaC(p) value for ANS binding indicates a specific interface with a significant hydrophobic component in the protein-ligand complex. The negatively charged sulphonate group of the anionic ligand is apparently not a major determinant of its binding. Phe-222 contributes to the binding affinity for ANS and the hydrophobicity of the binding site.

Dynamics of ANS Binding to Tuna Apomyoglobin Measured with Fluorescence Correlation Spectroscopy

The dynamics of the binding reaction of ANS to native and partly folded (molten globule) tuna and horse apomyoglobins has been investigated by fluorescence correlation spectroscopy and frequency domain fluorometry. The reaction rate has been measured as a function of apomyoglobin and ANS concentrations, pH, and temperature. Examination of the autocorrelation functions shows that the reaction rate is fast enough to be observed in tuna apomyoglobin, whereas the reaction rate in horse apomyoglobin is on the same time scale as diffusion through the volume or longer. Specifically, for tuna apomyoglobin at pH 7 and room temperature the on rate is 2200 μM −1 s −1 and the off rate is 5900 s −1, in comparison with k on = 640 μM −1 s −1 and k off = 560 s −1 for horse myoglobin as measured previously. The independence of the reaction rate from the ANS concentration indicates that the reaction rate is dominated by the off rate. The temperature dependence of the on-rate shows that this rate is diffusion limited. The temperature dependence of the off rates analyzed by Arrhenius and Ferry models indicates that the off rate depends on the dynamics of the protein. The differences between horse and tuna apomyoglobins in the ANS binding rate can be explained in terms of the three-dimensional apoprotein structures obtained by energy minimization after heme removal starting from crystallographic coordinates. The comparison of the calculated apomyoglobin surfaces shows a 15% smaller cavity for tuna apomyoglobin. Furthermore, a negative charge (D44) is present in the heme cavity of tuna apomyoglobin that could decrease the strength of ANS binding. At pH 5 the fluorescence lifetime distribution of ANS-apomyoglobin is bimodal, suggesting the presence of an additional binding site in the protein. The binding rates determined by FCS under these conditions show that the protein is either in the open configuration or is more flexible, making it much easier to bind. At pH 3, the protein is in a partially denatured state with multiple potential binding sites for ANS molecule, and the interpretation of the autocorrelation function is not possible by simple models. This conclusion is consistent with the broad distribution of ANS fluorescence lifetimes observed in frequency domain measurements.

8-anilino-1-naphthalene sulfonic acid (ANS) induces folding of acid unfolded cytochrome c to molten globule state as a result of electrostatic interactions.

Hydrophobic interaction of 8-anilino-1-naphthalene sulfonic acid (ANS) with proteins is one of the widely used methods for characterizing/detecting partially folded states of proteins. We have carried out a systematic investigation on the effect of ANS, a charged hydrophobic fluorescent dye, on structural properties of acid-unfolded horse heart cytochrome c at pH 2.0 by a combination of optical methods and electrospray ionization mass spectroscopy (ESI MS). ANS was found to induce, a secondary structure similar to native protein and quenching of fluorescence of tryptophan residue, in the acid-unfolded protein. However, the tertiary structure was found to be disrupted thus indicating that ANS stabilizes a molten globule state in acid-unfolded protein. To understand the mechanism of ANS-induced folding of acid-unfolded cytochrome c, comparative ESI MS, soret absorption, and tryptophan fluorescence studies using nile red, a neutral hydrophobic dye, and ANS were carried out. These studies suggested that, at low pH, electrostatic interactions between negatively charged ANS molecules and positively charged amino acid residues present in acid-unfolded cytochrome c are probably responsible for ANS-induced folding of acid-unfolded protein to partially folded compact state or molten globule state. This is the first experimental demonstration of ANS induced folding of unfolded protein and puts to question the usefulness of ANS for characterization/determination of partially folded intermediates of proteins observed under low pH conditions.

Effect of the hemolytic lectin CEL-III from Holothuroidea Cucumaria echinata on the ANS fluorescence responses in sensitive MDCK and resistant CHO cells.

The addition of CEL-III to sensitive MDCK cells preincubated with 8-anilino-1-naphthalenesulfonate (ANS) caused an increase in the fluorescence intensity of the probe. The increase in the ANS fluorescence caused by CEL-III was Ca2+-dependent and strongly inhibited by 0.1 M lactose, indicating that Ca2+-dependent binding of CEL-III to specific carbohydrate receptors on the plasma membrane is responsible for this phenomenon. In contrast, no significant effect of CEL-III on the ANS fluorescence was observed in CHO cells, which are highly resistant to CEL-III cytotoxicity. In MDCK cells, energy transfer from tryptophan residues to bound ANS molecules was observed in the presence of CEL-III, but not in CHO cells. Furthermore, the amount of ANS bound to MDCK cells increased as the concentration of CEL-III increased. Therefore, a simple interpretation is that the CEL-III-induced increase in ANS fluorescence is attributable to an increase of the hydrophobic region in the plasma membrane where ANS could bind. Immunoblotting analysis of proteins from cells treated with CEL-III indicated that CEL-III oligomers were irreversibly bound to the cells, and the amount of oligomer bound to MDCK cells was much greater than that bound to CHO cells under any conditions tested. The oligomerization may be accompanied by an enhancement of the hydrophobicity of CEL-III molecules, which in turn provides new ANS-binding sites. The difference in susceptibility of MDCK and CHO cells to CEL-III cytotoxicity may be due to a difference in oligomerization of bound CEL-III.