Passing of fluorescein derivatives into the hyphae of<Emphasis Type="Italic">Phanerochaete chrysosporium</Emphasis>

Fluorescein derivatives added into the growth medium were decolorized during submerged cultivation of Phanerochaete chrysosporium. The highest decrease of absorbance A450 was observed in the growth phase regardless of the presence of inducers Tween 80 or poly(ethylene glycol) (PEG). Fluorescein linked to PEG was prepared and, after addition to cultures, shown to stimulate the production of lignin peroxidase. Passing of fluorescing substances into hyphae (observed by confocal microscopy) showed that they were concentrated on some structures inside hyphae.     

New polymeric model substrates for the study of microbial ligninolysis.

Lignin model dimers are valuable tools for the elucidation of microbial ligninolytic mechanisms, but their low molecular weight (MW) makes them susceptible to nonligninolytic intracellular metabolism. To address this problem, we prepared lignin models in which unlabeled and alpha-14C-labeled beta-O-4-linked dimers were covalently attached to 8,000-MW polyethylene glycol (PEG) or to 45,000-MW polystyrene (PS). The water-soluble PEG-linked model was mineralized extensively in liquid medium and in solid wood cultures by the white rot fungus Phanerochaete chrysosporium, whereas the water-insoluble PS-linked model was not. Gel permeation chromatography showed that P. chrysosporium degraded the PEG-linked model by cleaving its lignin dimer substructure rather than its PEG moiety. C alpha-C beta cleavage was the major fate of the PEG-linked model after incubation with P. chrysosporium in vivo and also after oxidation with P. chrysosporium lignin peroxidase in vitro. The brown rot fungus Gloeophyllum trabeum, which unlike P. chrysosporium lacks a vigorous extracellular ligninolytic system, was unable to degrade the PEG-linked model efficiently. These results show that PEG-linked lignin models are a marked improvement over the low-MW models that have been used in the past.     

Spatially well-defined binary brushes of poly(ethylene glycol)s for micropatterning of active proteins on anti-fouling surfaces

We report a novel method for micropatterning of active proteins on anti-fouling surfaces via spatially well-defined and dense binary poly(ethylene glycol)s (PEGs) brushes with controllable protein-docking sites. Binary brushes of poly(poly(ethylene glycol) methacrylate-co-poly(ethylene glycol)methyl ether methacrylate), or P(PEGMA-co-PEGMEMA), and poly(poly(ethylene glycol)methyl ether methacrylate), or P(PEGMEMA), were prepared via consecutive surface-initiated atom transfer radical polymerizations (SI-ATRPs) from a resist-micropatterned Si(100) wafer surface. The terminal hydroxyl groups on the side chains of PEGMA units in the P(PEGMA-co-PEGMEMA) microdomains were activated directly by 1,1'-carbonyldiimidazole (CDI) for the covalent coupling of human immunoglobulin (IgG) (as a model active protein). The resulting IgG-coupled PEG microdomains interact only and specifically with target anti-IgG, while the other PEG microregions effectively prevent specific and non-specific protein fouling. When extended to other active biomolecules, microarrays for specific and non-specific analyte interactions with a high signal-to-noise ratio could be readily tailored.     

Interaction between proteins and polyphosphazene derivatives having a galactose moiety

For tissue engineering applications, it is necessary to balance the need for specific biological interactions with the need to prevent unfavorable nonspecific interactions. For this purpose, novel poly[(organo)phosphazenes] were synthesized having galactose and/or poly(ethylene glycol) (PEG) side chains. The synthesis was described previously. Here, we investigate the human serum albumin (HSA) adhesion to these polymers using surface plasmon resonance (SPR). We could conclude that the incorporation of PEG reduced the protein adsorption. The influence of the galactose moieties was investigated using SPR and a sugar-lectin binding assay. The interaction between a lectin (Peanut agglutinin, PNA or Ricinus communis-agglutinin, RCA) and the polyphosphazene derivatives was evaluated. Type IIA polymers, having aminohexyl-galactose, phenylalanine ethyl ester, and glycine ethyl ester side chains, were capable of binding with the lectin. As the amount of galactose was increased, the extent of the galactose specific lectin binding was also increased (higher RU or absorbance). PEG containing polymers failed to bind specifically with the lectin. The presence of PEG, either as a spacer or as additional chains, interfered with the establishment of contact between the galactose and the binding site on the lectin. The adsorption of PNA or RCA to these types of polymers was attributed to nonspecific interactions. SPR was also used to determine rate and equilibrium constants. In addition the effect of the addition of water soluble polyphosphazenes on the enzymatic cleavage of o-nitrophenyl-beta-D-galactopyranoside was investigated. The galactose moieties were not available as inhibitors because of the presence of PEG.     

Polymerization of pentachlorophenol and ferulic acid by fungal extracellular lignin-degrading enzymes.

High-molecular-weight polymers were produced by a crude concentrated supernatant from ligninolytic Phanerochaete chrysosporium cultures in a reaction mixture containing pentachlorophenol and a humic acid precursor (ferulic acid) in the presence of a detergent and H2O2. Pure manganese peroxidase, lignin peroxidase, and laccase were also shown to catalyze the reaction.     

Enzymatic nanoreactors for environmentally benign biotransformations. 1. Formation and catalytic activity of supramolecular complexes of laccase and linear-dendritic block copolymers

We describe the construction of enzymatic nanoreactors through noncovalent envelopment of a glycoprotein by amphiphilic linear-dendritic AB or ABA copolymers. The synthetic procedure is based on the regioselective adsorption of dendritic poly(benzyl ether)-block-linear poly(ethylene glycol)-block-dendritic poly(benzyl ether) or linear poly(ethylene oxide)-block-dendritic poly(benzyl ether) copolymers onto the oxidative enzyme laccase from Trametes versicolor in aqueous medium. The complexes formed have improved catalytic activity compared with the native enzyme (77-85 nkat/mL vs 60 nkat/mL, respectively) and are more stable at elevated temperatures up to 70 degrees C. Experiments with deglycosylated laccase confirm that the glycoside fragments in the native enzyme serve as the anchor sites for the linear-dendritic copolymers. The enzymatic nanoreactors are able to effectively oxidize series of substrates: phenolic compounds (syringaldazine) and hydrophobic polyaromatic hydrocarbons (anthracene and benzo[a]pyrene) under "green" chemistry conditions.     

Ethylene production from alpha-oxo-gamma-methylthiobutyric acid is a sensitive measure of ligninolytic activity by Phanerochaete chrysosporium.

Production of hydroxyl radical in ligninolytic cultures was determined by measuring the alpha-oxo-gamma-methylthiobutyric acid-dependent production of ethylene gas. The results showed that the pattern of ethylene production was very similar to that of ligninolytic activity [[14C]lignin leads to 14CO2). Furthermore, nutritional parameters, which are known to affect ligninolytic activity, affected OH.-radical-dependent ethylene production in a similar fashion. The results indicate that assay for ethylene production from alpha-oxo-gamma-methylthiobutyric acid is a simple and sensitive measure of ligninolytic activity by Phanerochaete chrysosporium.     

Synthesis and characterization of novel poly[(organo)phosphazenes] with cell-adhesive side groups

There is a need to develop new scaffold materials with controlled surface properties for tissue engineering applications. For that purpose novel biodegradable poly[(organo)phosphazenes] were synthesized. A cell-binding molecule, galactose, was introduced via a spacer, either 6-aminohexanol (AH) or poly(ethylene glycol) (PEG; M(w) = 3400). Some polymers were substituted with an additional PEG chain of different molecular weights (M(w) = 750 or 5000). The polyphosphazene derivatives were characterized by 1H NMR. T(g) and T(m) were determined using differential scanning calorimetry. A detailed surface analysis of the polymers using X-ray photoelectron spectroscopy (XPS), secondary ion mass spectroscopy (SIMS), and dynamic contact angle (DCA) measurements was performed. Typical backbone and side chain fragments were detected by SIMS and confirmed the polymer composition. Compared to that of the reference polymer (having only amino acid ester side groups), an increased value of the specific ether carbon groups from PEG confirmed the enrichment of PEG at the surface of PEG-Gal polymers. However, the values were lower than expected. DCA studies showed that the galactose moieties were present at the surface after exposure to an aqueous environment. XPS results confirmed the similarity between experimental and theoretical values for the AH-Gal polymers. This indicated the presence of galactose moieties at the surface, which was confirmed by the DCA data because the contact angles were low compared to those of the other polymers.     

Aqueous polymer two-phase systems formed by new thermoseparating polymers

A set of new polymers that can be used as phase forming components in aqueous two-phase systems is presented. All polymers studied have thermoseparating properties i.e. form one separate polymer enriched phase and one aqueous solution when heated above the critical temperature. This property makes the polymers attractive alternatives to the polymers used in traditional aqueous two-phase systems such as poly(ethylene glycol) (PEG) and dextran. The thermal phase separation simplifies recycling of the polymers, thus making the aqueous two-phase systems more cost efficient and suitable for use in large scale. Thermoseparating polymers studied have been copolymers of ethylene oxide and propylene oxide (EO-PO), poly (N-isopropylacrylamide) (poly-NIPAM), poly vinyl caprolactam (poly-VCL) and copolymers of N-isopropylacrylamide and vinyl caprolactam with vinyl imidazole (poly(NIPAM-VI) and poly(VCL-VI), respectively). In addition, the copolymer poly(NIPAM-VI) has the property to be uncharged at pH above 7.0 and positively charged at lower pH. This allows the partitioning of protein to be directed by changing the pH in the system instead of the traditional addition of salt to direct the partitioning. Hydrophobically modified EO-PO copolymer (HM-(EO-PO)) with alkyl groups (C₁₄) at both ends forms two-phase system with for example poly(NIPAM-VI). The phase diagram for poly(NIPAM-VI)/HM-(EO-PO) was determined and the model proteins lysozyme and BSA were partitioned in this system. For BSA in poly(NIPAM-VI)/HM-(EO-PO) system a change in pH from 8.0 to 5.4 results in a change of partition coefficient from K=0.8 to K=5.1, i.e. BSA could be transferred from the HM-(EO-PO) phase to the poly(NIPAM-VI) phase. BSA partitioning in poly(NIPAM-VI)/HM-(EO-PO) system allows quantitative BSA recovery, and recoveries of poly(NIPAM-VI) and HM-(EO-PO) were 53% and 92%, respectively, after the thermoseparation step.     

Synthesis of biocompatible PEG-Based star polymers with cationic and degradable core for siRNA delivery

Star polymers with poly(ethylene glycol) (PEG) arms and a degradable cationic core were synthesized by the atom transfer radical copolymerization (ATRP) of poly(ethylene glycol) methyl ether methacrylate macromonomer (PEGMA), 2-(dimethylamino)ethyl methacrylate (DMAEMA), and a disulfide dimethacrylate (cross-linker, SS) via an "arm-first" approach. The star polymers had a diameter ~15 nm and were degraded under redox conditions by glutathione treatment into individual polymeric chains due to cleavage of the disulfide cross-linker, as confirmed by dynamic light scattering. The star polymers were cultured with mouse calvarial preosteoblast-like cells, embryonic day 1, subclone 4 (MC3T3-E1.4) to determine biocompatibility. Data suggest star polymers were biocompatible, with ≥ 80% cell viability after 48 h of incubation even at high concentration (800 μg/mL). Zeta potential values varied with N/P ratio confirming complexation with siRNA. Successful cellular uptake of the star polymers in MC3T3-E1.4 cells was observed by confocal microscopy and flow cytometry after 24 h of incubation.     

Mechanism of the positive effect of poly(ethylene glycol) addition in enzymatic hydrolysis of steam pretreated lignocelluloses

The efficiency of enzymatic hydrolysis of lignocellulses can be increased by addition of surfactants and polymers, such as poly(ethylene glycol) (PEG). The effect of PEG addition on the cellulase adsorption was tested on various steam pretreated lignocellulose substrates (spruce, willow, hemp, corn stover, wheat straw, sweet sorghum bagasse). A positive effect of PEG addition was observed, as protein adsorption has decreased and free enzyme activities (FP, β-glucosidase) have increased due to the additive. However, the degree of enhancement differed among the substrates, being highest on steam pretreated spruce. Results of lignin analysis (pyrolysis-GC/MS, (31)P?NMR) suggest that the effect of PEG addition is in connection with the amount of unsubstituted phenolic hydroxyl groups of lignin in the substrate. Adsorption experiments using two commercial enzyme preparations, Celluclast 1.5L (Trichoderma reesei cellulase) and Novozym 188 (Aspergillus niger β-glucosidase) suggested that enzyme origins affected on the adsorptivity of β-glucosidases.     

A search for ligninolytic peroxidases in the fungus pleurotus eryngii involving alpha-keto-gamma-thiomethylbutyric acid and lignin model dimers

Because there is some controversy concerning the ligninolytic enzymes produced by Pleurotus species, ethylene release from alpha-keto-gamma-thiomethylbutyric acid (KTBA), as described previously for Phanerochaete chrysosporium lignin peroxidase (LiP), was used to assess the oxidative power of Pleurotus eryngii cultures and extracellular proteins. Lignin model dimers were used to confirm the ligninolytic capabilities of enzymes isolated from liquid and solid-state fermentation (SSF) cultures. Three proteins that oxidized KTBA in the presence of veratryl alcohol and H2O2 were identified (two proteins were found in liquid cultures, and one protein was found in SSF cultures). These proteins are versatile peroxidases that act on Mn2+, as well as on simple phenols and veratryl alcohol. The two peroxidases obtained from the liquid culture were able to degrade a nonphenolic beta-O-4 dimer, yielding veratraldehyde, as well as a phenolic dimer which is not efficiently oxidized by P. chrysosporium peroxidases. The former reaction is characteristic of LiP. The third KTBA-oxidizing peroxidase oxidized only the phenolic dimer (in the presence of Mn2+). Finally, a fourth Mn2+-oxidizing peroxidase was identified in the SSF cultures on the basis of its ability to oxidize KTBA in the presence of Mn2+. This enzyme is related to the Mn-dependent peroxidase of P. chrysosporium because it did not exhibit activity with veratryl alcohol and Mn-independent activity with dimers. These results show that P. eryngii produces three types of peroxidases that have the ability to oxidize lignin but lacks a typical LiP. Similar enzymes (in terms of N-terminal sequence and catalytic properties) are produced by other Pleurotus species. Some structural aspects of P. eryngii peroxidases related to the catalytic properties are discussed.     

Influence of electrostatic interactions on partitioning in aqueous polyethylene glycol/dextran biphasic systems: Part I

To study the influence of charges on the partition of solutes in aqueous two-phase systems of polyethylene glycol and dextran, partition coefficients of dimethylaminoethyl-dextran, trimethylamino-dextran, and bis (alpha,omega)-amino-poly(ethylene glycol) were determined as a function of pH (range 2 to 12) and ionic strength. These polymers are derivatives of the phase forming components and carry ionizable groups that are charged or uncharged depending on the pH. Unexpectedly, the largest differences in the partition coefficients were found at high pH, where the modified polymers are uncharged. In addition, the partitioning of low-molecular-weight model compounds, ethylenediamine and iminodiacetic acid, as well as poly-L-lysine and poly(allylamine) was analyzed. A consistent pattern was observed in the partition of polyelectrolytes reflecting the influence of charge, but another property of aqueous phase systems unrelated to charge and changing with pH seems to be superimposed. (c) 1995 John Wiley & Sons, Inc.     

Biosynthesis of poly(3-hydroxybutyrate) copolymers by Azotobacter chroococcum 7B: A precursor feeding strategy

A precursor feeding strategy for effective biopolymer producer strain Azotobacter chroococcum 7B was used to synthesize various poly(3-hydroxybutyrate) (PHB) copolymers. We performed experiments on biosynthesis of PHB copolymers by A. chroococcum 7B using various precursors: sucrose as the primary carbon source, various carboxylic acids and ethylene glycol (EG) derivatives [diethylene glycol (DEG), triethylene glycol (TEG), poly(ethylene glycol) (PEG) 300, PEG 400, PEG 1000] as additional carbon sources. We analyzed strain growth parameters including biomass and polymer yields as well as molecular weight and monomer composition of produced copolymers. We demonstrated that A. chroococcum 7B was able to synthesize copolymers using carboxylic acids with the length less than linear 6C, including poly(3-hydroxybutyrate-co-3-hydroxy-4-methylvalerate) (PHB-4MHV) using Y-shaped 6C 3-methylvaleric acid as precursor as well as EG-containing copolymers: PHB–DEG, PHB–TEG, PHB–PEG, and PHB–HV–PEG copolymers using short-chain PEGs (with n?≤?9) as precursors. It was shown that use of the additional carbon sources caused inhibition of cell growth, decrease in polymer yields, fall in polymer molecular weight, decrease in 3-hydroxyvalerate content in produced PHB–HV–PEG copolymer, and change in bacterial cells morphology that were depended on the nature of the precursors (carboxylic acids or EG derivatives) and the timing of its addition to the growth medium.     

Photocurable biodegradable liquid copolymers: synthesis of acrylate-end-capped trimethylene carbonate-based prepolymers, photocuring, and hydrolysis

Various photocurable liquid biodegradable trimethylene carbonate (TMC)-based (co)oligomers were prepared by ring-opening (co)polymerization of TMC with or without L-lactide (LL) using low molecular weight poly(ethylene glycol) (PEG) (mol wt 200, 600, or 1000) or trimethylolpropane (TMP) as an initiator. Resultant (co)oligomers were pastes, viscous liquids, or liquids at room temperature, depending on the monomer composition and monomer/initiator ratio. Liquid (co)oligomers were subsequently end-capped with acrylate groups. Upon visible-light irradiation in the presence of camphorquinone as a radical generator, rapid liquid-to-solid transformation occurred to produce photocured solid. The photocuring yield increased with photoirradiation time, photointensity, and camphorquinone concentration. The photocured polymers derived from low molecular weight PEG (PEG200) and TMP exhibited much reduced hydrolysis potential compared with PEG1000-derived polymers in terms of weight loss, water uptake, and swelling depth. Force-distance curve measurements by nanoindentation using atomic force microscopy clearly showed that Young's moduli of the photocured polymer films decreased with increasing hydrolysis time. Their potential biomedical applications are discussed.     

Synthesis of polyamidoamine dendrimers having poly(ethylene glycol) grafts and their ability to encapsulate anticancer drugs

Polyamidoamine dendrimers having poly(ethylene glycol) grafts were designed as a novel drug carrier which possesses an interior for the encapsulation of drugs and a biocompatible surface. Poly(ethylene glycol) monomethyl ether with the average molecular weight of 550 or 2000 was combined to essentially every chain end of the dendrimer of the third or fourth generation via urethane bond. The poly(ethylene glycol)-attached dendrimers encapsulating anticancer drugs, adriamycin and methotrexate, were prepared by extraction with chloroform from mixtures of the poly(ethylene glycol)-attached dendrimers and varying amounts of the drugs. Their ability to encapsulate these drugs increased with increasing dendrimer generation and chain length of poly(ethylene glycol) grafts. Among the poly(ethylene glycol)-attached dendrimers prepared, the highest ability was achieved by the dendrimer of the fourth generation having the poly(ethylene glycol) grafts with the average molecular weight of 2000, which could retain 6.5 adriamycin molecules or 26 methotrexate molecules/dendrimer molecule. The methotrexate-loaded poly(ethylene glycol)-attached dendrimers released the drug slowly in an aqueous solution of low ionic strength. However, in isotonic solutions, methotrexate and adriamycin were readily released from the poly(ethylene glycol)-attached dendrimers.     

Acid-sensitive polyethylene glycol conjugates of doxorubicin: preparation, in vitro efficacy and intracellular distribution

Coupling anticancer drugs to synthetic polymers is a promising approach of enhancing the antitumor efficacy and reducing the side-effects of these agents. Doxorubicin maleimide derivatives containing an amide or acid-sensitive hydrazone linker were therefore coupled to alpha-methoxy-poly(ethylene glycol)-thiopropionic acid amide (MW 20000 Da), alpha,omega-bis-thiopropionic acid amide poly(ethylene glycol) (MW 20000 Da) or alpha-tert-butoxy-poly(ethylene glycol)-thiopropionic acid amide (MW 70000 Da) and the resulting polyethylene glycol (PEG) conjugates isolated through size-exclusion chromatography. The polymer drug derivatives were designed as to release doxorubicin inside the tumor cell by acid-cleavage of the hydrazone bond after uptake of the conjugate by endocytosis. The acid-sensitive PEG conjugates containing the carboxylic hydrazone bonds exhibited in vitro activity against human BXF T24 bladder carcinoma and LXFL 529L lung cancer cells with IC70 values in the range 0.02-1.5 microm (cell culture assay: propidium iodide fluorescence or colony forming assay). In contrast, PEG doxorubicin conjugates containing an amide bond between the drug and the polymer showed no in vitro activity. Fluorescence microscopy studies in LXFL 529 lung cancer cells revealed that free doxorubicin accumulates in the cell nucleus whereas doxorubicin of the acid-sensitive PEG doxorubicin conjugates is primarily localized in the cytoplasm. Nevertheless, the acid-sensitive PEG doxorubicin conjugates retain their ability to bind to calf thymus DNA as shown by fluorescence and visible spectroscopy studies. Results regarding the effect of an acid-sensitive PEG conjugate of molecular weight 20000 in the chorioallantoic membrane (CAM) assay indicate that this conjugate is significantly less embryotoxic than free doxorubicin although antiangiogenic effects were not observed.     

Enzymatic preparation of streptavidin-immobilized hydrogel using a phenolated linear poly(ethylene glycol)

Hybrid gels constructed from proteins and polymers have attracted a wide range of attention in the field of biomedicine and bioengineering. We report herein the enzymatic preparation of polymer–protein hybrid hydrogels composed of terminally bis-functionalized linear poly(ethylene glycol) (PEG) and streptavidin (SA). PEG was conjugated with tyramine to introduce terminal phenolic hydroxyl (Ph-OH) groups. A peptide tag containing a tyrosine residue (G₄Y-tag) was genetically introduced at the C-terminus of SA. The Ph-OH-modified PEG and G₄Y-tagged SA (SA-G₄Y) were treated by horseradish peroxidase (HRP) in the presence of hydrogen peroxide (H₂O₂) to yield (PEG-Ph-OH)–(SA-G₄Y) hybrid gels. Biotinylated enhanced green fluorescent protein (biotin-EGFP) was selectively captured in the obtained hybrid gels, indicating that SA-G₄Y retained its biological function. The amount of biotin-EGFP immobilized in the hybrid gels depended on the concentration of SA-G₄Y. In addition, biotinylated bacterial alkaline phosphatase (biotin-BAP) was immobilized in the hybrid gel. The immobilized biotin-BAP exhibited more than 95% of the initial activity after 5 rounds of recycling. The results suggest the facile functionalization of the hybrid gel with a variety of biotinylated functional molecules.     

Comparison of Hydrolysis Efficiency and Enzyme Adsorption of Three Different Cellulosic Materials in the Presence of Poly(ethylene Glycol)

The addition of non-ionic surfactants has recently been confirmed to positively affect the enzymatic hydrolysis of cellulosic materials. However, the functional mechanisms of these surfactants remain unclear. This work investigated the influence of poly(ethylene glycol) (PEG) on the enzymatic hydrolysis of three cellulosic materials, namely, acid steam-exploded corn straw, pure microcrystalline cellulose (Avicel PH101), and bagasse sulfite pulp (BSP). The results showed that PEG addition led to varied effects on the enzymatic hydrolysis of different cellulosic materials. Addition of PEG was most effective on the enzymatic hydrolysis of PH101 and weakly effective on the hydrolysis of BSP. We further investigated PEG concentrations and enzymatic activities in the supernatant during hydrolysis and found that the positive effects of PEG treatment might contribute to its influence on enzyme desorption from different substrates. We also found that the efficiency of PEG depended on its capacity to bind to different substrates. PEG exhibited stronger affinity to pure cellulose than to the two other lignocellulosic substrates. These findings are helpful in further revealing the mechanism of surfactants and improving the enzymatic hydrolysis process.     

Mechanism of poly(ethylene glycol) interaction with proteins

Poly(ethylene glycol) (PEG) is one of the most useful protein salting-out agents. In this study, it has been shown that the salting-out effectiveness of PEG can be explained by the large unfavorable free energy of its interaction with proteins. Preferential interaction measurements of beta-lactoglobulin with poly(ethylene glycols) with molecular weights between 200 and 1000 showed preferential hydration of the protein for those with Mr greater than or equal to 400, the degree of hydration increasing with the increase in poly(ethylene glycol) molecular weight. The preferential interaction parameter had a strong cosolvent concentration dependence, with poly(ethylene glycol) 1000 having the sharpest decrease with an increase in concentration. The preferential hydration extrapolated to zero cosolvent concentration increased almost linearly with increasing size of the additive, suggesting steric exclusion as the major factor responsible for the preferential hydration. The poly(ethylene glycol) concentration dependence of the preferential interactions could be explained in terms of the nonideality of poly(ethylene glycol) solutions. All the poly(ethylene glycols) studied, when used at levels of 10-30%, decreased the thermal stability of beta-lactoglobulin, suggesting that caution must be exercised in the use of this additive at extreme conditions such as high temperature.