Influence of binding of sodium dodecyl sulfate, all-trans-retinol, palmitate, and 8-anilino-1-naphthalenesulfonate on the heat-induced unfolding and aggregation of beta-lactoglobulin B

Heat treatment of bovine beta-lactoglobulin B (beta-LG) causes it to partially unfold and aggregate via hydrophobic association and intra- and interprotein disulfide bonds. The first stage, which involves a "loosening" of the native structure, is influenced by the environmental conditions, such as pressure, pH, and added solutes. In the present study, four potential beta-LG ligands [palmitate, sodium dodecyl sulfate (SDS), 8-anilino-1-naphthalenesulfonate (ANS), and all-trans-retinol (retinol)] were added to beta-LG solutions prior to heat treatment for 12 min at temperatures between 40 and 93 degrees C. The extent of the changes in secondary and tertiary structures, unfolding, and aggregation at 20 degrees C were determined by circular dichroism, fluorescence, and alkaline- and SDS-polyacrylamide gel electrophoresis (PAGE). Both palmitate and SDS stabilized the native structure of beta-LG against heat-induced structural flexibility, subsequent unfolding, and denaturation. Retinol was less effective, probably because of its lower affinity for the calyx-binding site, and ANS did not stabilize beta-LG, suggesting that ANS did not bind strongly in the calyx. It was also noted that holding a beta-LG solution with added SDS or ANS promoted the formation of a hydrophobically associated non-native dimer.     

Influence of binding of sodium dodecyl sulfate, all-trans-retinol, and 8-anilino-1-naphthalenesulfonate on the high-pressure-induced unfolding and aggregation of beta-lactoglobulin B

Bovine beta-lactoglobulin B (beta-LG) is susceptible to pressure treatment, which unfolds it, allowing thiol-catalyzed disulfide bond interchange to occur, facilitating intermolecular bonding (both noncovalent and disulfide). In the present study, beta-LG was mixed with sodium dodecyl sulfate (SDS), all-trans-retinol (retinol), or 8-anilino-1-naphthalenesulfonate (ANS) on a 1:1.1 molar basis, and aliquots were held at pressures between 50 and 800 MPa for 30 min at pH 7.2 and 20 degrees C. Polyacrylamide gel electrophoresis (PAGE) showed that beta-LG alone (control) was converted into a non-native monomer and a series of dimers, trimers, etc., at pressures beyond 100 MPa; SDS inhibited the formation of non-native species up to 200 MPa, and neither retinol nor ANS inhibited the formation of the non-native species as effectively as SDS. At pressures beyond 350 MPa, SDS ceased to have any inhibitory effect, but both ANS and retinol showed significant inhibition. The near- and far-UV CD patterns and the ANS fluorescent data were consistent with the PAGE data, but the retinol fluorescent data did not show sufficient change to interpret. The results suggested that there were three discernible structural stages. In Stage I (0.1-150 MPa), the native structure is stable; in Stage II (200-450 MPa), the native monomer is reversibly interchanging with non-native monomers and disulfide-bonded dimers; and in Stage III (>500 MPa), the free CysH in non-native monomer and dimer interacts with -S-S- bonds to produce high molecular weight aggregates of beta-LG. SDS inhibited the Stage I to Stage II transition at 200 MPa, and ANS and retinol inhibited the Stage II to Stage III transition at 600 MPa.     

Effectiveness of the Iodide Ligand Along with two Surfactants on Desorbing Heavy Metals from Soils

Used with one of two surfactants (SDS, an anionic surfactant, and Triton X-100, a nonionic surfactant), the ligand, I^? was evaluated as a washing agent for the desorption of Cd from naturally and artificially contaminated soils. Increasing amounts of the ligand, I^?, with a surfactant, specifically removes higher levels of Cd but not Cu, Zn and Pb. After seven washings, the ligand, I^? with the nonionic surfactant, Triton X-100, removed 65 and 90% of the Cd from soils I and II, containing respectively, to 15 and 1275 mg of Cd/kg. The ligand, I^?, and the anionic surfactant, SDS, removed 35 and 70% of the Cd from soils I and II, respectively. Before washing, the carbonate fraction of soil I contained the most Cd (48%) while the exchangeable and carbonate fractions of soil II contained 29 and 33% of the total Cd, respectively. For soil I, SDS with/ without the ligand desorbed Cd mainly from the carbonate and oxide fractions, while only Triton X-100 with ligand I^? could remove Cd from the exchangeable fraction. For soil II, Cd was desorbed from the exchangeable fraction only when either surfactant was used in combination with the ligand. Thus, a surfactant with ligand can extract specific heavy metals from soils and selective sequential extraction is useful in identifying which fraction can be targeted by the surfactant – ligand agent.     

塿土对阴-非离子表面活性剂的吸附特征研究

以平衡吸附法研究了塿土对阴离子表面活性剂（SDS）、非离子表面活性剂（TritonX-100、Tween80和Brij35）的吸附特征,考察了pH、阴-非离子表面活性剂混合对塿土吸附表面活性剂的影响。结果表明,非离子表面活性剂在塿土上吸附等温线均呈L型,且均符合Freundlich和Langmuir方程;塿土对SDS的吸附等温线呈LS型,可用Freundlich方程来描述;塿土对4种表面活性剂吸附量的大小顺序为Tween80〉SDS〉Brij35〉TritonX-100。当阴-非离子表面活性剂一起进入土壤中,SDS-Brij35之间的相互影响不大;TritonX-100与SDS相互作用较大,无论二者以何种方式混合都会使TritonX-100在塿土上的吸附量增加,SDS的吸附量下降;SDS与Tween80之间的相互作用最大,混合后吸附量均下降,但Tween80吸附量降低的幅度最大。pH对非离子表面活性剂的吸附影响不大,而随着pH的增加,塿土对SDS的吸附百分率明显下降;在pH为8.0时,塿土对非离子表面活性剂的吸附百分率达到80以上。因此在选择合适的表面活性剂进行有机污染土壤修复和治理时,考虑土壤的特性和表面活性剂的结构是非常重要的。     

Toxicity of Synthetic Surfactants to the Marine Macroalga, Ulva lactuca

The toxicities of three synthetic surfactants to the marine macroalga, Ulva lactuca, have been examined by monitoring chlorophyll a fluorescence quenching. The anionic surfactant, sodium dodecyl sulphate (SDS), exerted no measurable toxicity over the concentration range 0?C10 mg L^?1, while presence of the non-ionic surfactant, Triton X-100 (TX), elicited a small reduction in photochemical efficiency that was independent of concentration. The cationic surfactant, hexadecyltrimethylammonium bromide (HDTMA), incurred a dose-dependent response to ??3 mg L^?1 (EC₅₀?=?2.4 mg L^?1), but a reduction in toxicity thereafter. Presence of TX had little effect on the toxicity of HDTMA but an equimolar concentration of SDS directly offset the impact of HDTMA on photochemical efficiency. Relative toxicities of the surfactants are attributed to differences in affinity for the algal surface and tendencies to disrupt cell membranes and interact with intracellular macromolecules. Non-linear dose responses and antagonistic effects are attributed to non-specific interactions between molecules of the same surfactant and electrostatic interactions between molecules of different amphiphilic character.     

Pressure-induced unfolding and aggregation of the proteins in whey protein concentrate solutions

Whey protein concentrate solutions (12% w/v, pH 6.65 +/- 0.05) were pressure treated at 800 MPa for 20-120 min and then examined using size exclusion chromatography (SEC), small deformation rheology, transmission electron microscopy, and various types of one-dimensional (1D) and two-dimensional (2D) polyacrylamide gel electrophoresis (PAGE). The pressure-treated samples showed a time-dependent loss of native whey proteins by SEC and 1D PAGE and a corresponding increase in non-native proteins and protein aggregates of different sizes. These aggregates altered the viscosity and opacity of the samples and were shown to be cross-linked by intermolecular disulfide bonds and by noncovalent interactions using 1D PAGE [alkaline (or native), sodium dodecyl sulfate (SDS), and SDS of reduced samples (SDS(R))] and 2D PAGE (native:SDS and SDS:SDS(R)). The sensitivity of the major whey proteins to pressure was in the order beta-lactoglobulin B (beta-LG B) > beta-LG A > bovine serum albumin (BSA) > alpha-lactalbumin (alpha-LA), and the large internal hydrophobic cavity of beta-LG may have been partially responsible for its sensitivity to high-pressure treatments. It seemed likely that, at 800 MPa, the formation of a beta-LG disulfide-bonded network preceded the formation of disulfide bonds between alpha-LA or BSA and beta-LG to form multiprotein aggregates, possibly because the disulfide bonds of alpha-LA and BSA are less exposed than those of beta-LG either during or after pressure treatment. It may be possible that intermolecular disulfide bond formation occurred at high pressure and that hydrophobic association became important after the high-pressure treatment.     

Effect of physicochemical conditions on peptide-peptide interactions in a tryptic hydrolysate of beta-lactoglobulin and identification of aggregating peptides

The objective of this study was to characterize the changes in peptide solubility resulting from changing some physicochemical conditions in a tryptic hydrolysate of beta-lactoglobulin (beta-LG). The turbidity (500 nm) of a 1% solution of tryptic peptides was measured at pH 3-10, at 5, 25, and 50 degrees C, in the presence of different salt concentrations (0, 0.5, and 1 M NaCl), in the presence of denaturing and reducing agents (6 M urea, 5% SDS, or 5% beta-mercaptoethanol), and under an electric field (isoelectric focusing). The results reveal an increase in turbidity of the peptide solution at pH 4, but a slight increase in turbidity was also observed at pH 8, which is attributable to peptides linked by disulfide bridges. The effect of temperature and ionic strength on the turbidity occurring at pH 4 indicates that mainly hydrophobic interactions are involved in the aggregation process. The material in the precipitate at pH 4 was identified as the peptides beta-LG 1-8, 15-20, and 41-60 and non-hydrolyzed alpha-lactalbumin. These results suggest that a limited number of peptides are involved in the aggregation process observed at pH 4, some of which having bioactive (beta-LG 15-20, ACE inhibitor, and opioid) or emulsifying properties (beta-LG 41-60). Aggregation of these peptides at acidic pH indicates that a simple acidification step could represent an easy process for isolating peptidic fractions enriched in bioactive or functional peptides.     

Ethanol effect on the structure of beta-lactoglobulin b and its ligand binding

The changes of structure and ligand binding properties of beta-LG B have been studied by fluorescence and circular dichroism spectroscopy in ethanolic solutions. Fluorescence measurements of retinol/beta-LG interactions at 480 nm in various ethanol concentrations show that the maximal fluorescence intensity induced by this interaction between retinol and beta-LG is observed around 20% v/v of ethanol. It is reduced to zero at 40% and 50% of ethanol. These results suggest that there are two distinct structural changes in beta-LG occurring between 20% and 30% and around 40% of ethanol. The first transition, which increases affinity and the apparent number of binding sites for retinol, may be related or similar to the Tanford transition. The strong quenching of retinol emission at 480 nm in 40% of ethanol indicates the radical transformation of beta-LG tertiary structure and the release of retinol. CD spectra at the aromatic region show that secondary and tertiary structures of beta-LG are not significantly affected between 0% and 20% of ethanol. In 30% of ethanol, beta-sheet percentage of beta-LG decreases with respect to native beta-LG (from 55% to 46%). beta-Sheet percentage in beta-LG increases in 40% and 50% alcohol (51% and 53%) relative to 30% of ethanol, which also indicates the strong rearrangement of the secondary structure of beta-LG, while its tertiary structure and beta-LG interactions are radically changed.     

Hydrophobic probe binding of beta-lactoglobulin in the native and molten globule state induced by high pressure as affected by pH,KIO(3) and N-ethylmaleimide

High hydrostatic pressure (HHP) at 500 MPa and 50 degrees C induces beta-LG into the molten globule state. Retinol, cis-parinaric acid (CPA), and 1-anilino-naphthalene-8-sulfonate (ANS) fluorescence from pH 2.5 to 10.5 in the presence of the native and molten globule states of beta-LG indicate that retinol binds to beta-LG in the calyx, CPA at the surface hydrophobic site, and ANS in multiple hydrophobic sites. HHP treatment results in a decrease of beta-LG affinity for retinol and CPA, suggesting conformational changes in the calyx and surface hydrophobic site of beta-LG during HHP treatment. beta-LG treated by HHP in the presence of N-ethylmaleimide (NEM) retains retinol affinity, suggesting that NEM protects the calyx conformation of beta-LG during HHP treatment. HHP treatment of beta-LG in the presence of KIO(3) exhibits a great decrease of CPA affinity compared to HHP-treated beta-LG in the absence of KIO(3), suggesting the formation of non-native disulfide bonding at the CPA binding site.     

Pentachlorophenol interactions with soil

Pentachlorophenol (PCP) adsorption and desorption equilibrium was studied with two Menfro silt loam soils — upper horizon and lower horizon. For the adsorption studies the variables were: temperature (10 and 30 °C) and the amount of organic matter. The variables for the desorption studies were: temperature (10 and 30 °C), pH and the presence of an anionic and a cationic surfactant. The results from these studies confirmed the importance of soil organic matter for adsorption of PCP on the soils. The adsorption data at different temperatures indicated the physical nature of the adsorption process. The desorption data produced non-singularity and some PCP was irreversibly adsorbed onto the soil despite repeated washings. Increased pH increased the desorption of PCP from the soil. The anionic surfactant, sodium dodecylbenzene sulfonate (SDS) was able to desorb significant amounts of PCP from the soil at doses equal to critical micelle concentration (CMC). But, the nonionic surfactant, surfactant, Triton X-405 required a much higher dose (twice the CMC) to cause a significant desorption of PCP from the soil.     

表面活性剂溶液清洗油污土壤试验研究

选用两种阴离子型表面活性剂十二烷基苯磺酸钠(LAS)和十二烷基硫酸钠(SDS)及两种非离子型表面活性剂Triton X-100和Tween 80,研究了临界胶束浓度(CMC)附近各表面活性剂溶液对柴油的增溶及一次性清洗油污土壤的能力。结果表明:在CMC附近,各表面活性剂对柴油的增溶能力大小顺序为SDSSDS>Triton X-100>Tween 80,两种阴离子型表面活性剂的清洗效果优于两种非离子型表面活性剂。     

Influence of rhamnolipids and triton X-100 on the biodegradation of three pesticides in aqueous phase and soil slurries.

The effect of surfactants on the biodegradation of trifluralin and atrazine (by Streptomyces PS1/5) and coumaphos (by degrading consortia from a contaminated cattle dip) in liquid cultures and soil slurries was tested at different concentrations of a rhamnolipid mixture (Rh-mix) and Triton X-100 (TX-100). The extent of trifluralin biodegradation in liquid culture was improved at high concentrations of both surfactants. The extent of atrazine degradation dropped in the presence of either surfactant. Coumaphos biodegradation improved slightly at Rh-mix dosages >3000 microM; however, it was readily inhibited by TX-100 at amounts above the critical micelle concentration. In soil slurries, the extent of both trifluralin and atrazine biodegradation was higher in Hagerstown A (HTA) soil than in Hagerstown B (HTB) soil and was not significantly affected by the presence of either surfactant. The onset of trifluralin biodegradation was retarded at higher concentrations of surfactants. In the absence of surfactant, up to 98% of coumaphos in both soil slurries was transformed. At increasing dosages of Rh-mix, the onset of coumaphos biodegradation was retarded, but the removal efficiency of the pesticide increased. Rh-mix and TX-100 depletion was observed during Streptomyces PS1/5 growth in liquid cultures. Rh-mix concentration also decreased during coumaphos biodegradation, whereas TX-100 concentration was not affected. These results suggest that surfactants, added for the purpose of increasing the apparent water solubility of hydrophobic organic compounds, may have unintended effects on both the rate and extent of biodegradation of the target compounds if the surfactants can also be degraded by the microorganisms in the system.     

Interactions between bovine beta-lactoglobulin and peptides under different physicochemical conditions

The aim of this study was to determine if peptides could interact with beta-lactoglobulin (beta-LG) and what the physicochemical conditions promoting their interaction with the protein are. The binding of negatively charged (beta-LG 125-135 and 130-135), positively charged (beta-LG 69-83 and 146-149), and hydrophobic (alphaS1-CN 23-34 and beta-LG 102-105, both bioactive peptides) peptides to bovine beta-LG was determined using an ultrafiltration method under different physicochemical conditions: pH 3.0, 6.8, and 8.0; buffers of 0.05 and 0.1 M; 4, 25, and 40 degrees C; beta-LG/peptide ratios of 1:5 and 1:10. At pH 3.0, none of the peptides interacted with beta-LG at any temperature, buffer molarity, or beta-LG/peptide ratio probably due to electrostatic repulsions between the highly protonated species. At pH 6.8 and 8.0, charged peptides beta-LG 130-135, 69-83, and 146-149 bound to beta-LG under some physicochemical conditions, possibly by nonspecific binding. However, both hydrophobic peptides probably bind to the inner cavity (beta-barrel) of beta-LG, provoking the release of materials absorbing at 214 nm. Given the known biological activities of the hydrophobic peptides used in this study (opioid and ACE-inhibitory activities), their binding to beta-LG may be relevant to a better understanding of the physiological function of the protein.     

Surfactant-Enhanced Removal of Cu (II) and Zn (II) from a Contaminated Sandy Soil

Batch tests were conducted to know the effectiveness of using surfactants only and surfactants with a complexing agent to remove Cu (II) and Zn (II) from an artificially contaminated sandy soil. SDS (sodium dodecyl sulfate), AOT (alpha-olefin sulfonate) and Tx-100 (Triton X-100) were the surfactants selected as the washing liquids. Complexing agent EDTA (ethylenediaminetetraacetic acid) was also selected for washing the soil. To avoid external factors from interfering with the cleaning process, artificial soil formed by a mixture of clean sand and bentonite was used to form contaminated soil samples. The amount of organic matter present was insignificant. Compared to extraction by distilled water, tests indicated that a six-fold increase in copper extraction occurred due to the presence of surfactants and/or the complexing agent EDTA. Compared to extraction by distilled water, zinc extraction by surfactants and or the complexing agent EDTA was nearly 1.2 to 1.3 times more. Effects of competition as well as interference associated with the adsorption and desorption of these metals are also very briefly reported.     

Reduced immunogenicity of beta-lactoglobulin by conjugation with carboxymethyl dextran differing in molecular weight

To reduce the immunogenicity of beta-lactoglobulin (beta-LG), two beta-LG-carboxymethyl dextran (CMD) conjugates (Conj. 40 and Conj. 162) were prepared by using water-soluble carbodiimide (EDC). The molar ratios of beta-LG to CMD in Conj. 40 and Conj. 162 were 8:1 and 7:1, respectively. Each conjugate maintained approximately 50% of the retinol binding activity of beta-LG. Structural analyses by intrinsic fluorescence, CD spectra, and ELISA with monoclonal antibodies indicated that the surface of beta-LG in each conjugate was covered by CMD without great disruption of native conformation. By conjugation with CMD, the antibody response to beta-LG was reduced in BALB/c, C3H/He, and C57BL/6 mice, which was eminent in Conj. 162. The results of B cell epitope scanning using overlapping synthesized peptides showed that the linear epitope profiles of the conjugates were similar to those of beta-LG, whereas the antibody response to each epitope was reduced, which was eminent in Conj. 162. It was concluded that conjugation with CMD of higher molecular weight is effective in reducing the immunogenicity of beta-LG and that masking of epitopes by CMD is responsible for the reduced immunogenicity.     

Effect of milk concentration on the irreversible thermal denaturation and disulfide aggregation of beta-lactoglobulin

The kinetics of beta-lactoglobulin (beta-LG) denaturation in reconstituted skim milk samples of various concentrations (9.6-38.4% total solids) over a wide temperature range (75-100 degrees C) was studied. The thermal denaturation of beta-LG had a reaction order of 1.5 at all milk solids concentrations and at all temperatures. The rate of denaturation of beta-LG was markedly dependent on the milk solids concentration and the heating temperature. At 75 degrees C, the thermal denaturation of beta-LG was retarded at higher milk solids concentrations. However, this retardation was less pronounced at higher temperatures so that a similar rate of denaturation was observed at all milk solids concentrations at 100 degrees C. From an examination of the level of disulfide-aggregated beta-LG, it was evident that most, but not all, of the denatured beta-LG was involved in disulfide-aggregated complexes, either with other denatured whey proteins or with the casein micelles. As with beta-LG denaturation, the rate of disulfide aggregation of beta-LG was markedly dependent on the milk solids concentration.     

Cholesterol solubilization in aqueous micellar solutions of quillaja saponin, bile salts, or nonionic surfactants

Quillaja saponin in aqueous solution enhanced cholesterol solubility by as much as a factor of 10(3) at room temperature. Increased temperature and [NaCl] increased cholesterol solubility, whereas solubility was greatest at an aqueous pH of 4.6 at 298 K. Although various saponin sources were observed to differ in their abilities to solubilize cholesterol, trends in their solubilization properties with changing aqueous phase parameters were consistent. Surfactant molecules containing fused-ring structures as their hydrophobic portion, such as sodium cholate, sodium deoxycholate, and quillaja saponin, solubilized cholesterol significantly better than the linear hydrocarbon chain surfactants Tween 20 and Triton X-100. Mixtures of surfactants studied were found to exhibit synergistic effects: they formed micelles at lower concentrations than did those formed by the individual surfactants themselves, and they had a better ability to solubilize cholesterol. The knowledge obtained from these studies improves our understanding of cholesterol association with saponin and other types of surfactants and enhances the potential for using saponins for the solubilization and extraction of hydrophobic solutes in various pharmacological and industrial applications.     

Reduced immunogenicity of beta-lactoglobulin by conjugation with acidic oligosaccharides

Bovine beta-lactoglobulin (beta-LG) was conjugated with the acidic oligosaccharides, alginic acid oligosaccharide (ALGO) and phosphoryl oligosaccharides (POs) by the Maillard reaction to reduce the immunogenicity of beta-LG. The molar ratios of beta-LG to ALGO and POs in the conjugates were 1:6 and 1:8. The carbohydrate-binding sites in the beta-LG-ALGO conjugate were partially identified to be (60)Lys, (77)Lys, (100)Lys, (138)Lys, and (141)Lys. The isoelectric point of each conjugate was lower than that of beta-LG. CD spectra indicated that the secondary structure of beta-LG was almost maintained after conjugation. The results of fluorescence studies indicated that the conformation around Trp had not changed in each conjugate and that the surface of each conjugate was covered with a saccharide chain. Structural analyses with monoclonal antibodies indicated that the conformation around (8)Lys-(19)Trp (beta-sheet, random coil, short helix) in the conjugates had changed, whereas the native structure was maintained around (15)Val-(29)Ile (beta-sheet) and (125)Thr-(135)Lys (alpha-helix). The beta-LG-ALGO and beta-LG-POs conjugates maintained 77 and 70% of the retinol binding activity of beta-LG. Conjugation with ALGO and POs substantially enhanced the thermal stability of beta-LG. The anti-beta-LG antibody response was markedly reduced after immunization with both conjugates in BALB/c, C57BL/6, and C3H/He mice. B cell epitopes of beta-LG and the conjugate recognized in these mice were determined with 15-mer multipin peptides, and the linear epitope profiles of the conjugates were found to be similar to those of beta-LG, whereas the antibody response to each epitope was dramatically reduced. In particular, effective reduction of the antibody response was observed in the vicinity of the carbohydrate-binding sites. Conjugation of beta-LG with these acidic oligosaccharides was effective in reducing the immunogenicity of beta-LG. The conjugates obtained in this study are edible, so they would be very useful for food application.     

Modification of bovine beta-lactoglobulin by glycation in a powdered state or in an aqueous solution: effect on association behavior and protein conformation

The effect of glycation with lactose on the association behavior and conformational state of bovine beta-lactoglobulin (beta-LG) was studied, using size exclusion chromatography, polyacrylamide gel electrophoresis, proteolytic susceptibility, and binding of a fluorescent probe. Two modification treatments were used, i.e., aqueous solution glycation and dry-way glycation. The results showed that the latter treatment did not significantly alter the nativelike behavior of the protein while the former treatment led to important structural changes. These changes resulted in a specific denatured beta-LG monomer, which covalently associated via the free thiol group. The homodimers thus formed and the expanded monomers underwent subsequent aggregation into a high molecular weight species, via noncovalent interactions. The association behavior of glycated beta-LG is discussed with respect to the known multistep denaturation/aggregation process of nonmodified beta-LG.     

Heat treatment of beta-lactoglobulin: structural changes studied by partitioning and fluorescence

Functional properties of whey protein concentrates (WPC) are primarily dependent on the degree of denaturation of beta-lactoglobulin (beta-LG), the major globular whey protein. Irreversible modifications in the tertiary structure and association state of beta-LG after heat treatment were studied by partition in aqueous two-phase systems and fluorescence quenching. Partitioning of preheated beta-LG in two-phase systems containing 5% (w/w) poly(ethylene glycol) and 7% (w/w) dextran, between pH 6.0 and7.0, are appropriately related with the intensity of heat treatment. An increase in the partition coefficient of beta-LG was observed with increasing temperature of heat treatment. On the other hand, fluorescence quenching of beta-LG by acrylamide was used to study the conformational flexibility of the protein at pH values between 4. 0 and 9.0. The values of bimolecular quenching rate constant (k(q)) obtained showed that beta-LG appears to be more flexible at high pH values, while at low pH the protein assumes a more compact form. The efficiency of acrylamide quenching on preheated beta-LG was substantially more pronounced than for the untreated protein. This difference can be ascribed to the presence of unfolded monomers and aggregates of denatured molecules formed after heat treatment, whose tryptophanyl residues are more exposed to the solvent. In conclusion, the results suggest that partition studies in aqueous two-phase systems and fluorescence quenching are very useful tools to detect changes in conformation and aggregation of beta-LG induced by heat treatment.