Effect of glycosylation on the partition behavior of a human antibody in aqueous two-phase systems.

Human proteins are expressed in some hosts wrongly glycosylated or nonglycosylated. Although it is accepted that glycosylation contributes to the stability of the protein in solution, the effect of glycosylation on the stability of human antibodies is not fully understood. In this work, we present solubility studies of two human antibodies that have the same primary structure but different glycosylation pattern. The studies were done by monitoring the partitioning behavior of both proteins in a series of aqueous two-phase systems at and away the isoelectric point of the proteins and at different temperatures. Our studies show that in the absence of direct electrostatic forces, the partitioning behavior of the antibodies depends on the presence or absence of the polysaccharide chains. Overall, the nonglycosylated protein is less soluble than the glycosylated one. The potential of aqueous two-phase systems for the separation of the glycosylated and nonglycosylated proteins was also explored. A simple series of extractions seems to be enough to separate the glycosylated variety from the nonglycosylated one at high purity but low yields.

Purification of human antibodies from transgenic corn using aqueous two-phase systems.

A recombinant human antibody expressed in corn was purified using aqueous two-phase extraction. The antibody was an immunoglobulin G fully unglycosylated. Using systems of different compositions and/or pHs in each of one or two partitioning stages followed by one more stage in which the antibody was precipitated at the liquid/liquid interface facilitated the removal of different impurities in each stage. The best system yields a product 72% pure (22-fold purification) with a yield of 49%. The optimum extraction was done in two partitioning stages followed by an interfacial precipitation stage using poly(ethylene)glycol/potassium phosphate systems. NaCl was added to the first stage to eliminate large molecular weight impurities. The pH in the first stage was kept at 6 but a pH of 8 was used in the second stage and in the precipitation stage.

Cold cataracts: a naturally occurring aqueous two-phase system.

The cytoplasm of the eye lenses shows a liquid-liquid phase transition similar to the one observed in aqueous two-phase systems. This phenomenon is known as cold cataracts. We have studied the solution behavior of the main protein fractions that constitute the lenses' cytoplasm using small-angle neutron scattering and dynamic light scattering. Our results provide evidence that an intricate balance of forces underlines the physical phenomena responsible for the optical properties of the lenses and for the phase transition that is observed as the temperature is lowered below some critical value. These forces include solvent-mediated forces besides the more conventional Coulombic and dispersion forces. This study suggests that solvent mediated forces must be included to successfully model liquid-liquid phase transitions like the ones observed in cold cataracts or in aqueous two-phase systems.

Effects of Cosolvents and pH on Protein Adsorption on Polystyrene Latex: A Dynamic Light Scattering Study.

Dynamic light scattering was used to study the adsorption of two proteins with different surface properties (IgG and HSA) on negatively charged polystyrene latex. The proteins were adsorbed from water and from water/methanol and water/glycerol mixtures at various pH. Some striking differences between the adsorption behaviors of the proteins were observed. Whereas the thickness of the adsorbed layer of HSA was extremely sensitive to pH and solvent composition, that of IgG was not. IgG mainly showed an end-on orientation on polystyrene whereas several different surface orientations are suggested for HSA under different conditions. The addition of methanol inhibited the adsorption of HSA on the latex, but it did not affect the adsorption of IgG. In contrast, the addition of glycerol increased the thickness of the adsorbed layers of both proteins. So, the orientation of IgG on the latex is insensitive to pH but is a function of the kind of solvent whereas both pH and solvent strongly affect the adsorption of HSA. This is a puzzling result since both cosolvents should equally affect the adsorption of both proteins if the dominant forces for adsorption are the same. Therefore, we concluded that, whereas hydrophobic interactions are the dominant force in the adsorption behavior of HSA, van der Waals forces are the main forces involved in the attachment of IgG to the lattices. Copyright 2000 Academic Press.

Accumulation of lead by Anabaena cylindrica : mathematical modeling and an energy dispersive X-ray study.

The ability of microorganisms to selectively adsorb various heavy metal ions has been recognized for over a decade. We have investigated the biosorption of lead by an active culture of the cyanobacterium Anabaena cylindrica. Energy dispersive X-ray microanalysis was used to evaluate the different uptake mechanisms in the various subcellular regions. Three were identified: a very fast adsorption mechanism in the cell envelope; a time-dependent deposition reaction on the cell surface; and an adsorption mechanism, also time dependent, on the polyphosphate body inside the cell. Atomic absorption spectrometry was then used to quantify the changes with time of bulk fluid concentrations of lead solutions exposed to cyanobacteria. A mass transfer kinetic model was developed which quantitatively predicts the concentration of lead in cells of Anabaena cylindrica as a function of spatial dimensions and time. The model predictions are consistent with a pattern, documented in literature and confirmed by our own experimental evidence, of a very fast uptake in the cell envelope and then a longer uptake period inside the cell. Our experimental evidence also revealed a time-dependent uptake mechanism on the surface of the cells, which is included in the model. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 408-418, 1997.

A small-angle neutron scattering study of gamma-crystallins near their isoelectric point.

In this paper, a small-angle neutron scattering study of gammaII-crystallins near their isoelectric point is presented. The experiments were carried out using protein concentrations of 5.7-85.7 mg/ml at temperatures in the range 11 -60 degrees C. The experimental data were analyzed using an ellipsoidal model for intraparticle correlations and the mean spherical approximation for interparticle correlations. Our studies revealed that gammaII-crystallins have a thick hydration layer, which is possibly due to the special arrangement of polar and ionic groups on their surface. The temperature scan shows that, as a result of relatively strong attractive forces, clusters of two, three, or higher oligomers are present below 20 degrees C. Our results suggest that protein clusters, with a distinctive hydration layer, form a protein-rich phase that separates from a protein-lean phase as the temperature is decreased below some threshold value.

Protein partitioning at the isoelectric point: influence of polymer molecular weight and concentration and protein size.

We report the partition coefficient, K(p') at the isoelectric point of lysozyme, chymotrypsinogen A, albumin, transferrin, and catalase in 64 different polyethylene(PEG)/ dextran(Dx)/water systems. We study the trends of the partition coefficient with protein type, polymer concentration, and polymer molecular weight. We find that the partition coefficient decreases with increasing tie line length for lysozyme, albumin, transferrin, and catalase for which K(p) is less than 1, but increases for chymotrysinogen for which K(p) is larger than 1. The effect of the tie line length on the partition coefficient is larger for the large proteins than for the small proteins. The partition coefficient decreases with increasing protein molecular weight except for lysozyme suggesting that lysozyme is present as a dimer or a trimer. The partition coefficient decreases with increasing PEG molecular weight, but the magnitude of the increase is larger for the smaller PEG molecular eights and tends to level of at high PEG molecular weight. The partition coefficient increases with increasing dextran (Dx) molecular weight for chymotrypsinogen but decreases for catalase. The partition coefficients of lysozyme, albumin, and transferrin increase with increasing Dx molecular weight from Dx 10(4) to Dx 1.1 x 10(5) and then slightly decrease from Dx 1.1 x 10(5) to Dx 5 x 10(5). The experimental results are analyzed using a statistical thermodynamics model. The experimental results are analyzed using a statistical thermodynamics model. The experiments suggest that protein partitioning at the isoelectric point in aqueous two-phase systems is strongly related to the size of the proteins and polymers. Finally, the impossibility of obtaining data completely independent of polymer concentration is emphasized.

Analysis of polymer molecular weight distributions in aqueous two-phase systems.

The partitioning of proteins and other biomaterials between two aqueous phases containing polyethyleneglycol and dextran is a strong function of the molecular weight of the two polymers. Although both polymers are polydispersed (especially Dx) most theoretical treatments refer only to the average molecular weight (number or mass) and assume that the molecular weight distribution of each polymer is the same in both phases. In this work the molecular weight distribution of each polymer is the same in both phases. In this work the molecular weight distributions of four stock solutions of PEG (4000, 6000, 10,000 and 20,000) and four stock solutions of Dx (10,000, 40,000, 110,000 and 500,000) were measured using High Performance Gel Chromatography. The measurements were repeated on the phases formed by the polymer solutions after they were mixed and allowed to equilibrate. The molecular weight distribution of the Dx differed in the top and bottom phase; both differed from that of the stock solution. Although we believe that the molecular weight distribution for PEG also differs in the top and bottom phases, we were unable to determine this within the resolution of our instruments.

Temperature dependence of the partition coefficient of proteins in aqueous two-phase systems.

We report the partition coefficients of lysozyme, chymotrypsinogen-A, albumin and catalase in sixty four Polyethyleneglycol/Dextran/Water systems at 4, 25 and 40 degrees C. We found that the partition coefficients of the four proteins generally increase with increasing temperature. The influence of temperature on the partition coefficient seems to be highly dependent on the kind of protein which is partitioned and on the total polymer concentration, but does not, in general, depend on the molecular weight of the polymers. The partition coefficients of small and hydrophilic proteins like lysozyme and chymotrypsinogen-A are only slightly affected by changes in temperature, while the partition coefficients of bigger and more hydrophobic proteins like albumin and catalase are strongly affected by changes in temperature. The results suggest the incorporation of attractive forces (possible electrostatic) into a model previously reported by us.

Investigation of affinity partition chromatography using formate dehydrogenase as a model.

The enzyme formate dehydrogenase (FDH) was purified from the crude extract of Candida boidinii by affinity partition chromatography. The partition coefficient, K, of the enzyme was selectively increased by adding polyethylene glycol-Procion Red HE3b as an affinity ligand to the mobile phase in the chromatographic column. The increased K value led to early elution of the enzyme-ligand complex and separated the target protein from the main peak of the contaminants.

[Acute respiratory obstruction caused by laryngo-tracheal candidiasis in a HIV-positive patient]. / Ostruzione respiratoria acuta da candidosi laringotracheale in soggetto HIV positivo.

The authors report a case presenting highly complex symptomatology. In fact, when the patient came under observation he had had a cough, dyspnea, dysphagia and dysphonia for approximately three months. The biopsy, taken by direct laryngoscopy, indicated the presence of candidiasis in the subglottic and tracheal areas. Laboratory tests indicated complete anergy and patient tested serum positive to HIV. During hospitalization acute dyspnea arose requiring emergency tracheostomy.