Capillary electrophoretic separation of high-molecular-weight poly(ethylene glycol)-modified proteins

This study was designed to demonstrate the utility of capillary electrophoresis (CE) for separating high-molecular-weight poly(ethylene glycol) (PEG)-conjugated proteins. As a CE method, sodium dodecyl sulfate-capillary gel electrophoresis (SDS-CGE) was applied to analyze interferon alpha (IFN) modified with branched and trimer-structured PEG molecules. Five mono-PEG-IFN conjugates prepared with two branched PEGs (MW 20 and 40 kDa) and three trimer-structured PEGs (MW 23.5, 43.5, and 47 kDa) were purified by cation-exchange chromatography and their masses were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The SDS-CGE method showed high separation capacity by differentiating PEG-IFN conjugates with small differences in molecular size, such as PEG_40K-, PEG_43.5K-, and PEG_47K-IFNs, and it was useful for checking the purity of each mono-PEG-IFN. This study shows that SDS-CGE can well be utilized in the development and quality control of PEGylated proteins prepared with various types of PEG.     

Sites of modification of hemospan, a poly(ethylene glycol)-modified human hemoglobin for use as an oxygen therapeutic

Hemospan is an acellular hemoglobin-based oxygen therapeutic in clinical trials in Europe and the United States. The product is prepared by site-specific conjugation of maleimide-activated poly(ethylene) glycol (PEG, MW approximately 5500) to human oxyhemoglobin through maleimidation reactions either (1) directly to reactive Cys thiols or (2) at surface Lys groups following thiolation using 2-iminothiolane. The thiolation/maleimidation reactions lead to the addition of approximately 8 PEGs per hemoglobin tetramer. Identification of PEG modified globins by SDS-PAGE and MALDI-TOF reveals a small percentage of protein migrating at the position for unmodified globin chains and the remaining as separate bands representing globin chains conjugated with 1 to 4 PEGs per chain. Identification of PEG modification sites on individual alpha and beta globins was made using reverse-phase HPLC, showing a series of alpha globins conjugated with 0 to 3 PEGs and a series of beta globins conjugated with 0 to 4 PEGs per globin. Mass analysis of tryptic peptides from hemoglobin thiolated and maleimidated with N-ethyl maleimide showed the same potential sites of modification regardless of thiolation reaction ratio, with seven sites identified on beta globins at beta8, beta17, beta59, beta66, beta93, beta95, and beta132 and three sites identified on alpha globins at alpha7, alpha16, and alpha40.     

Mechanism of polymer-induced hemolysis: nanosized pore formation and osmotic lysis

Hemolysis induced by antimicrobial polymers was examined to gain an understanding of the mechanism of polymer toxicity to human cells. A series of cationic amphiphilic methacrylate random copolymers containing primary ammonium groups as the cationic functionality and either butyl or methyl groups as hydrophobic side chains have been prepared by radical copolymerization. Polymers with 0-47 mol % methyl groups in the side chains, relative to the total number of monomeric units, showed antimicrobial activity but no hemolysis. The polymers with 65 mol % methyl groups or 27 mol % butyl groups displayed both antimicrobial and hemolytic activity. These polymers induced leakage of the fluorescent dye calcein trapped in human red blood cells (RBCs), exhibiting the same dose-response curves as for hemoglobin leakage. The percentage of disappeared RBCs after hemolysis increased in direct proportion to the hemolysis percentage, indicating complete release of hemoglobin from fractions of RBCs (all-or-none leakage) rather than partial release from all cells (graded leakage). An osmoprotection assay using poly(ethylene glycol)s (PEGs) as osmolytes indicated that the PEGs with MW > 600 provided protection against hemolysis while low molecular weight PEGs and sucrose had no significant effect on the hemolytic activity of polymers. Accordingly, we propose the mechanism of polymer-induced hemolysis is that the polymers produce nanosized pores in the cell membranes of RBCs, causing an influx of small solutes into the cells and leading to colloid-osmotic lysis.     

High-performance liquid chromatographic determination of PEG 600 in human urine

Polyethylene glycols (PEGs) are non-ionic, water-soluble synthetic polymers which have been widely used for many applications. Since they are of very low toxicity and are readily excreted in urine, PEGs in the molecular weight range 400–6000 have been used extensively in the study of intestinal physiology in man. A high-performance liquid chromatographic (HPLC) method has been developed for the determination of PEG 600 in human urine, which includes a pre-column derivatisation step. The dibenzoate derivatives of PEG 600 can be quantitatively prepared, and this, coupled with ultraviolet detection at 230 nm, has greatly improved the limit of detection for the determination of PEGs by HPLC. A suitable extraction procedure has also been developed which enabled PEG levels in urine to be monitored with much greater sensitivity than any previously reported method.     

Removal of peroxides in polyethylene glycols by vacuum drying: Implications in the stability of biotech and pharmaceutical formulations

The purpose of this study was to investigate the utility of vacuum drying for removing peroxides from polyethylene glycols (PEGs). PEG solutions (PEG 1450 and PEG 20000) containing varying levels of peroxides were prepared by storing under different light and temperature conditions. PEGs containing low and high levels of peroxides were vacuum dried from dilute and concentrated solutions (2.5%, 7.5%, 15%, and 50% wt/vol of PEG 1450 and 2.5%, 7.5%, 15%, and 25% wt/vol of PEG 20000). Ferrous ion oxidation in presence of ferric ion indicator xylenol orange (FOX) colorimetric assay was used to determine the concentration of peroxides. Peroxide content in PEGs increased upon storage. The increase was more pronounced when PEGs were stored at higher temperatures and exposed to light. Vacuum drying at 0.1 mm Hg for 48 hours at 25°C resulted in greater than 90% decrease in the level of peroxides in all cases except when high peroxide containing 25% wt/vol solution of PEG 20000 or 50% wt/vol solution of PEG 1450 were dried. The reduction in the level of peroxides for PEGs dried from high peroxide containing 25% wt/vol solution of PEG 2000 and 50% wt/vol solution of PEG 1450 was found to be 88% and 52%, respectively. Oxidation of methionine in Met-Leu-Phe peptide was significantly reduced when vacuum-dried PEGs were used. Vacuum drying PEG solutions at low pressures is an effective method for the removal of the residual peroxides present in commercially available PEGs. Published: July 28, 2006     

Partially folded intermediate state of concanavalin A retains its carbohydrate specificity

A systematic investigation of the effect of polyethylene glycol (PEG) 200 and 400 on the solution conformation of concanavalin A (con A) was made using circular dichroism (CD), tryptophan fluorescence, 1-anilino-8-naphthalenesulfonic acid (ANS) binding, and size-exclusion chromatography. Far-UV CD spectra of con A at 30%(v/v) PEGs show the retention of ordered secondary structure as compared to 70%(v/v) PEGs. Near-UV CD spectra showed the retention of native-like spectral features in the presence of 30%(v/v) PEGs. Intrinsic tryptophan fluorescence studies indicate a change in the environment of tryptophan residues on the addition of PEG. ANS binding was maximum at 30%(v/v) PEGs suggesting the compact "molten-globule"-like state with enhanced exposure of hydrophobic surface area. Size-exclusion chromatography indicates an intermediate hydrodynamic size at 30%(v/v) PEGs. GdnHCl denaturation of these states was a single-step, two-state transition. To study the possible minimum structural requirement in the specific binding, the effect of PEGs on the interaction of con A with ligand was investigated by turbidity measurements. The C50 value was less in PEG 400 suggesting the more inhibitory ability of PEG 400. The C50 value of PEGs was highest for dextran followed by glycogen, ovalbumin, and ovomucoid. From percentage inhibition of con A-ligands at 30%(v/v) PEG, maximum inhibition was in ovalbumin followed by ovomucoid, glycogen, and dextran. To summarize: con A at 30%(v/v) PEGs exists as compact intermediate with molten-globule-like characteristics, viz., enhanced hydrophobic surface area, retention of compact secondary as well as tertiary structure, and a considerable degree of carbohydrate binding specificity and activity. This result has significant implications on the molten globule state during the folding pathway(s) of proteins in general and quaternary association in the legume lectin in particular, where precise topology is required for their biological activities.     

Steric exclusion is the principal source of the preferential hydration of proteins in the presence of polyethylene glycols.

The preferential interactions of bovine serum albumin, lysozyme, chymotrypsinogen, ribonuclease A, and beta-lactoglobulin with polyethylene glycols (PEGs) of molecular weight 200-6,000 have been measured by dialysis equilibrium coupled with high precision densimetry. All the proteins were found to be preferentially hydrated in all the PEGs, and the magnitude of the preferential hydration increased with increasing PEG size for each protein. The change in the chemical potentials of the proteins with the addition of the PEGs had highly positive values, indicating a strong thermodynamic destabilization of the system by the PEGs. A viscosity study of the PEGs showed them to be randomly coiled polymers, as their radii of gyration were related to the molecular weight by Rg = aM0.55. The thickness of the effective shell impenetrable to PEG around protein molecules, calculated from the preferential hydration, was found to vary with PEG molecular weight in similar fashion as the PEG radius of gyration, supporting the proposal (Arakawa, T. & Timasheff, S.N., 1985a, Biochemistry 24, 6756-6762) that the preferential exclusion of PEGs from proteins is due principally to the steric exclusion of PEG from the protein domain, although favorable interactions with protein surface residues, in particular nonpolar ones, may compete with the exclusion. These thermodynamically unfavorable preferential exclusion interactions lead to the action of PEGs as precipitants, although they may destabilize protein structure at higher temperatures.     

Osmotic Pressure of Aqueous Polyethylene Glycols : Relationship between Molecular Weight and Vapor Pressure Deficit

Osmotic pressures (II) of aqueous solutions of polyethylene glycols (PEGs) of average relative molecular weight (M_r) between 200 and 10,000 were measured using vapor pressure deficit osmometry. The relationships between molarity and II were described with high precision by second order polynomials for each of the PEGs studied. In contrast to previous reports, equivalent weights of different polymers in solution did not generate the same II; low M_r PEGs generated a higher II than the higher M_r PEGs. The effect of PEGs upon II represents an interaction between concentration and M_r.     

Effect of polyethylene glycols on the function and structure of thiol proteases

Thiol proteases are industrially significant proteins with catalytic efficiency. The effect of low, medium and high molecular-weight poly (ethylene glycol) (PEG- 400, 6000 and 20000) on the stability of thiol proteases (papain, bromelain and chymopapain) has been studied by activity measurements using synthetic substrate. Structural studies performed on papain by far UV circular dichroism spectroscopic measurements indicate that there is loss in secondary structure of the protein in presence of increasing concentration of PEGs. Intrinsic fluorescence measurements lead us to conclude that tryptophan residues of protein encounter non-polar microenvironment in presence of PEG solvent while acrylamide quenching shows greater accessibility of tryptophan residues of papain in presence of PEGs. Extrinsic fluorescence measurements lead us to conclude that PEGs bind to the hydrophobic sites on the protein and thus destabilize it. Thermal denaturation studies show that melting temperature of papain is decreased in presence of PEGs. Possible mechanism of destabilization is discussed next. The results imply that caution must be exercised in the use of PEGs with thiol proteases or hydrophobic proteins in general, for different industrial applications, even at room temperature.     

Probing protein nanopores with poly(ethylene glycol)s

Neutral water-soluble poly(ethylene glycol)s (PEGs) have been extensively explored in protein nanopore research for the past several decades. The principal use of PEGs is to investigate the membrane protein ion channel physical characteristics and transport properties. In addition, protein nanopores are used to study polymer–protein interactions and polymer physicochemical properties. In this review, we focus on the biophysical studies on probing protein ion channels with PEGs, specifically on nanopore sizing by PEG partitioning. We discuss the fluctuation analysis of ion channel currents in response to the PEGs moving within their confined geometries. The advantages, limitations, and recent developments of the approach are also addressed.     

Wild tomato endosperm transcriptomes reveal common roles of genomic imprinting in both nuclear and cellular endosperm

Genomic imprinting is a conspicuous feature of the endosperm, a triploid tissue nurturing the embryo and synchronizing angiosperm seed development. An unknown subset of imprinted genes (IGs) is critical for successful seed development and should have highly conserved functions. Recent genome‐wide studies have found limited conservation of IGs among distantly related species, but there is a paucity of data from closely related lineages. Moreover, most studies focused on model plants with nuclear endosperm development, and comparisons with properties of IGs in cellular‐type endosperm development are lacking. Using laser‐assisted microdissection, we characterized parent‐specific expression in the cellular endosperm of three wild tomato lineages (Solanum section Lycopersicon). We identified 1025 candidate IGs and 167 with putative homologs previously identified as imprinted in distantly related taxa with nuclear‐type endosperm. Forty‐two maternally expressed genes (MEGs) and 17 paternally expressed genes (PEGs) exhibited conserved imprinting status across all three lineages, but differences in power to assess imprinted expression imply that the actual degree of conservation might be higher than that directly estimated (20.7% for PEGs and 10.4% for MEGs). Regardless, the level of shared imprinting status was higher for PEGs than for MEGs, indicating dissimilar evolutionary trajectories. Expression‐level data suggest distinct epigenetic modulation of MEGs and PEGs, and gene ontology analyses revealed MEGs and PEGs to be enriched for different functions. Importantly, our data provide evidence that MEGs and PEGs interact in modulating both gene expression and the endosperm cell cycle, and uncovered conserved cellular functions of IGs uniting taxa with cellular‐ and nuclear‐type endosperm.     

Biosynthesis,properties and potential of natural–synthetic hybrids of polyhydroxyalkanoates and polyethylene glycols

Chemical conjugation with poly(ethylene glycols) (PEGs) are established procedures to facilitate solubilisation of hydrophobic compounds. Such techniques for PEGylation have been applied to polyhydroxybutyrate. ‘BioPEGylation’ of such polyhydroxyalkanoates (PHAs) to form natural–synthetic hybrids has been demonstrated through the addition of PEGs to microbial cultivation systems. The strategic addition of certain PEGs not only supports hybrid synthesis but may also provide a technique for control of PHA composition and molecular mass, and by extension, their physico-mechanical properties. PHA composition and molecular mass control by PEGs is dependent upon the polyethers’ molecular mass, loading in the cultivation system, time of introduction and microbial species. Hybrid characterisation studies are in their infancy, but results to date suggest that PHA–PEG hybrids have subtle, but significant, differences in their physiochemical and material properties as a consequence of the PEGylation.     

Aggregation of human RBC in binary dextran-PEG polymer mixtures

The present study was prompted by prior reports suggesting that small polymers can affect RBC aggregation induced by large macromolecules. Human RBC were washed and re-suspended in isotonic buffer solutions containing 72.5 kDa dextran (DEX 70, 2 g/dl) or 35.0 kDa poly(ethylene glycol) (PEG 35, 0.35 g/dl), then tested for aggregation in these solutions with and without various concentrations of smaller dextrans (10.5 and 18.1 kDa) or PEGs (3.35, 7.5 and 10.0 kDa). RBC aggregation was measured at stasis and at low shear using a photometric cone-plate system (Myrenne Aggregometer) and RBC electrophoretic mobility (EPM) in the various polymer solutions via an automated system (E4, HaSoTec GmbH). Our results indicate: (1) a heterogeneous effect with greater reduction of aggregation for small PEGs added to DEX 70 or for small dextrans added to PEG 35 than for small polymers of the same species; (2) for cells in DEX 70, aggregation decreased with increasing molecular mass and concentration of the small dextrans or PEGs; (3) for cells in PEG 35, small dextrans decreased aggregation with increasing molecular mass and concentration, whereas small PEGs had minimal effects with a minor influence of concentration and an inverse association between molecular mass and inhibition of aggregation. RBC EPM results indicated the expected polymer depletion for cells in DEX 70 or PEG 35, and that small PEGs yielded greater EPM values than small dextrans for cells in PEG 35 whereas the opposite was true for cells in DEX 70. Interpretation of our results in terms of the depletion model for RBC aggregations appears appropriate, and our findings are consistent with the assumption that inhibition of aggregation occurs because of an increase of small molecules in the depletion region. Our results thus suggest the merit of further studies of red blood cell aggregation in binary polymer systems.     

Efficient synthesis of linear multifunctional poly(ethylene glycol) by copper(I)-catalyzed Huisgen 1,3-dipolar cycloaddition

Poly(ethylene glycol) (PEG) is a versatile biocompatible polymer. Improvement of its limited functionality (two chain termini) may significantly expand its current applications. In this communication, a simple and yet highly efficient strategy for the synthesis of linear multifunctional PEGs with "click" chemistry is reported. A short acetylene-terminated PEG was linked by 2,2-bis(azidomethyl)propane-1,3-diol using Cu(I)-catalyzed Huisgen 1,3-dipolar cycloaddition in water at room temperature. High-molecular-weight PEGs with pendant hydroxyl groups were obtained and characterized by 1H NMR and size-exclusion chromatography. A prototype bone-targeting polymeric drug delivery system was also successfully synthesized based on this new method. It demonstrates strong biomineral-binding ability and the ease of incorporating therapeutic agents into the delivery system. This simple "click" reaction approach provides a useful tool for the development of novel functional polymers and their conjugates for biomedical applications.     

Probing protein hydration and conformational states in solution.

The addition of polyethylene glycol (PEG), of various molecular weights, to solutions bathing yeast hexokinase increases the affinity of the enzyme for its substrate glucose. The results can be interpreted on the basis that PEG acts directly on the protein or indirectly through water activity. The nature of the effects suggests to us that PEG's action is indirect. Interpretation of the results as an osmotic effect yields a decrease in the number of water molecules, delta Nw, associated with the glucose binding reaction. delta Nw is the difference in the number of PEG-inaccessible water molecules between the glucose-bound and glucose-free conformations of hexokinase. At low PEG concentrations, delta Nw increases from 50 to 326 with increasing MW of the PEG from 300 to 1000, and then remains constant for MW-PEG up to 10,000. This suggests that up to MW 1000, solutes of increasing size are excluded from ever larger aqueous compartments around the protein. Three hundred and twenty-six waters is larger than is estimated from modeling solvent volumes around the crystal structures of the two hexokinase conformations. For PEGs of MW > 1000, delta Nw falls from 326 to about 25 waters with increasing PEG concentration, i.e., PEG alone appears to "dehydrate" the unbound conformation of hexokinase in solution. Remarkably, the osmotic work of this dehydration would be on the order of only one k T per hexokinase molecule. We conclude that under thermal fluctuations, hexokinase in solution has a conformational flexibility that explores a wide range of hydration states not seen in the crystal structure.     

The use of 1H-NMR spectroscopy and refractometry for investigation of the distribution of nonelectrolytes of N-alcohol series between human red blood cells and extracellular medium

Comparative analysis of 1H NMR spectroscopy and refractometry with respect to their application for investigating the distribution of nonelectrolytes of n-alcohol series (ethanol, 1,2-propanediol, glycerol) and polyethylene glycols (PEGs) with molecular masses of 400, 600, 1500 between human erythrocytes and extracellular medium was performed. The distribution coefficients (Q) for solutions of ethanol, 1,2-propanediol, glycerol, PEG-400, PEG-600 and PEG-1500 were obtained. The Q values decreased with the increase in the nonelectrolyte molecular mass from 1.23+/-0.12 for ethanol to 0.40+/-0.08 for PEG-1500 (1H NMR spectroscopy) and from 2.6+/-0.12 for ethanol to 0.23+/-0.03 for PEG-1500 (refractometry). It was shown that 1H-NMR high-resolution spectroscopy ensures more precise determination of Q values for nonelectrolytes with low molecular masses; for PEGs with high molecular masses, the accuracy of Q value calculation by this method was about 20%. On the contrary, refractometry can be used for investigating substances with high molecular masses; the error of Q value determination for solution of low-refractive substances, such as ethanol, may be more than 50%.     

Stabilization of chloroperoxidase by polyethylene glycols in aqueous media: kinetic studies and synthetic applications

Chloroperoxidase (CPO) is one of the most versatile of the heme peroxidase enzymes for synthetic applications. Despite the potential use of CPO, commercial processes have not been developed because of the low water solubility of many organic substrates of synthetic interest and the limited stability due to inactivation by H(2)O(2). CPO catalytic properties have been studied in aqueous solutions in the presence of short-chain poly(ethylene glycol)s (PEGs), and the sulfoxidation of thioanisole, as model substrate, has been investigated. The addition of PEGs allows a better substrate solubilization in the reaction mixture and the enzyme to retain more of its initial activity, with respect to pure buffer. Kinetic studies were performed to optimize the experimental conditions, and complete enantioselective conversion to the (R)-sulfoxide (ee = 99%) was observed in the presence of PEG 200 and tri(ethylene glycol). The relevant stabilization of chloroperoxidase due to the presence of PEGs allows the enzyme to convert the substrate with significant product yields even after 10 days, with a consequent increase in enzyme productivity. This is a promising result in view of industrial application of the enzyme.     

Simple method for sample temperature calibration in a 500 MHz NMR spectrometer useful for protein studies

The melting point of several poly(ethylene glycols) (PEGs) was used to calibrate the temperature above ambient with the separation of the hydroxyl and methylene peaks of ethylene glycol (EG) on a 500 MHz nuclear magnetic resonance (NMR) spectrometer. The calibration is almost identical to a calibration of the EG sample on a 90 MHz NMR spectrometer using a thermocouple. The equation accurately predicts the thermal denaturation midpoint of the protein, hen egg white lysozyme. It is concluded that in the absence of a small magnet, the calibration of an EG sample using the melting points of PEGs provides a simple temperature calibration, for larger superconducting magnets, useful for protein stability studies.     

High-resolution analysis of parent-of-origin allelic expression in the Arabidopsis Endosperm

Genomic imprinting is an epigenetic phenomenon leading to parent-of-origin specific differential expression of maternally and paternally inherited alleles. In plants, genomic imprinting has mainly been observed in the endosperm, an ephemeral triploid tissue derived after fertilization of the diploid central cell with a haploid sperm cell. In an effort to identify novel imprinted genes in Arabidopsis thaliana, we generated deep sequencing RNA profiles of F1 hybrid seeds derived after reciprocal crosses of Arabidopsis Col-0 and Bur-0 accessions. Using polymorphic sites to quantify allele-specific expression levels, we could identify more than 60 genes with potential parent-of-origin specific expression. By analyzing the distribution of DNA methylation and epigenetic marks established by Polycomb group (PcG) proteins using publicly available datasets, we suggest that for maternally expressed genes (MEGs) repression of the paternally inherited alleles largely depends on DNA methylation or PcG-mediated repression, whereas repression of the maternal alleles of paternally expressed genes (PEGs) predominantly depends on PcG proteins. While maternal alleles of MEGs are also targeted by PcG proteins, such targeting does not cause complete repression. Candidate MEGs and PEGs are enriched for cis-proximal transposons, suggesting that transposons might be a driving force for the evolution of imprinted genes in Arabidopsis. In addition, we find that MEGs and PEGs are significantly faster evolving when compared to other genes in the genome. In contrast to the predominant location of mammalian imprinted genes in clusters, cluster formation was only detected for few MEGs and PEGs, suggesting that clustering is not a major requirement for imprinted gene regulation in Arabidopsis.     

Effect of polyethyleneglycol (PEG) on spontaneous and polymyxin B-induced histamine release from rat peritoneal mast cells

Mast cells were isolated from the peritoneal cavity of rat and purified by centrifugation in a gradient of Percoll. The spontaneous and polymyxin B-induced release of histamine was studied after preincubation of the cells with polyethyleneglycols (PEGs) of different molecular weight (200-20,000 dalton) and with fatty acid derivatives of PEG 6000. It was found that very low concentrations (less than 0.1%) of PEG 6000 reduced the spontaneous and polymyxin B-induced release of histamine to a greater extent than the same concentration of bovine serum albumin. The inhibition increased with the size of the PEGs, but was little affected by the presence of fatty acid ligand bound to PEG.