Partition Of Macromolecules Research Articles

Chemical modification of holey carbon supports for cryogenic-electron microscopy

The structural determination of biological molecules has recently undergone a revolution with the Cryogenic-Electron Microscopy (cryo-EM) technique advances. However, a poor partitioning of macromolecules into the holes of holey carbon support grids frequently limits 3D structural determination by Single Particles Analysis through cryo-EM. This work present a method to deposit, on gold-coated carbon grids, a self-assembled monolayer whose surface properties can be controlled by chemical modification. There is a demonstration of this approach utility to maximize the grid wettability while drive partitioning lipossomes into the holes, thereby enabling better results using cryo-EM. Gold-coated grids with 2-mercaptoethanol yields a hydrophilic surface to holey carbon cryo-EM supports. The method is simple and accessible requiring commercially available items at relatively low costs. The acquired cryo-micrographs for the liposome sample prepared on the SAM grids confirm the success on the drive partitioning of the species into the holes. This approach may enable structure determination of macromolecular targets that suffer from high affinity for holey carbon support.

Partitioning of Macromolecules between Two Interconnected Spherical Cavities

Simulations of excluded volume macromolecular chain encased in two interconnected cavities reveal for weak confinement a strong preference for a conformation of macromolecule in one of the two cavities. For a symmetric twin-cavity system, a conformational transition from the single cavity occupation to the conformation bridging the two cavities is observed on increasing confinement of encased chain. For asymmetric system of cavities with weak confinement the partitioning of chain segments between the two cavities is governed by classical partitioning law in which partitioning free energy increases linearly with chain length. On increasing chain length (or stronger confinement), a maximum in this dependence is observed followed by a decrease. This behavior is explained by correlation between increasing confinement and increasing concentration of encased chain. The position of the maximum reflects an increasing hinderance of the macromolecule in the larger of the two cavities and the start of the population...

Accumulation of Macromolecules in Pores from Dilute Solutions in Poor Solvent

The phenomenon of spontaneous accumulation of macromolecules in pores from the dilute solutions of flexible polymers in poor solvent is studied using the density functional theory. It is shown that the partitioning of macromolecules between the bulk solution and slitlike pore, on whose walls the segments are predominantly adsorbed, depends on the ability of a semidilute polymer solution to wet the wall of a pore and on its size, as well as on the degree of undersaturation of bulk solution and temperature. Partitioning coefficient increases in a jumpwise manner when the surface first-order phase transition or capillary separation take place. It is revealed that the regularities of capillary separation of polymer solutions in a poor solvent are similar to those observed during the capillary condensation of unsaturated vapor. The applicability of the Kelvin equation to describe the conditions of capillary separation of flexible polymer solutions is analyzed.

Evaluation of liquid chromatography column retentivity using macromolecular probes: IV. Poly(ethylene glycol) bonded phase

Interaction properties of the novel HPLC silica gel–poly(ethylene glycol) (PEG) bonded phase were evaluated applying polymeric test substances, viz. polystyrenes, poly(methyl methacrylate)s, poly(ethylene oxide)s and poly(2-vinyl pyridine)s, and eluents of different polarities. Silanols on the silica gel surface are well shielded by the PEG phase, and silanophilic adsorption of macromolecules is suppressed in comparison with most silica C 18 bonded phases. The adsorption of solutes on the OH groups of the PEG phase seems to be low as well. The partition of macromolecules in favor of the PEG phase is inferior to that observed in case of the silica C 18 phases. The volume of the PEG bonded phase is small and it is supposed that the PEG chains assume flat conformation on the silica gel surface.

Concentration Effects in Partitioning of Macromolecules into Pores with Attractive Walls

The influence of polymer concentration on the partitioning of flexible macromolecules into adsorptive pores was examined by simulations in an open system under good solvent conditions. In dilute solutions, the partition coefficient K(ε,φ) is sensitive to small variations of both polymer−pore attraction strength ε and concentration φ, whereas in semidilute solutions both influences level off. At weak attraction below critical adsorption energy εc, the coefficient K increases with φ in a fashion that resembles the weak-to-strong penetration transition found for purely steric exclusion (ε = 0) but modified as if the width of pores effectively increased. At moderate attraction above the critical condition the increasing concentration brings about a dramatic drop in the value of K. Additionally, the K(φ) dependences in attractive pores were calculated by an approximate method based on expressions for chemical potentials, and the results were in good agreement with the simulation data. The critical adsorption energy εc for excluded volume chains was found to depend on concentration; the compensation point K = 1 was located at about φ = 0.012 irrespective of the pore width. The energy and entropy contributions to the free energy of confinement were calculated and the compensation of their values at the critical condition was demonstrated. Furthermore, it was shown that the effect of concentration in polymer partitioning with adsorptive pores can alternatively be regarded as a confined adsorption and two alternatives of the adsorbed amount Γ were calculated. The adsorption and depletion concentration profiles φI(x) in the pore in equilibrium with the bulk solution were presented and their variation with concentration φ and attraction ε was analyzed.

Cytoplasmic signals mediate apical early endosomal targeting of endotubin in MDCK cells.

Endotubin is an integral membrane protein that targets into apical endosomes in polarized epithelial cells. Although the role of cytoplasmic targeting signals as mediators of basolateral targeting and endocytosis is well established, it has been suggested that apical targeting requires either N-glycosylation of the ectoplasmic domains or partitioning of macromolecules into glycolipid-rich rafts. However, we have previously shown that the cytoplasmic portion of endotubin possesses signals that are necessary for its proper sorting into the apical early endosomes. To further define the targeting signals involved in this apically directed event, as well as to determine if the cytoplasmic domain was sufficient to mediate apical endosomal targeting, we generated a panel of endotubin and Tac-antigen chimeras and expressed them in Madin-Darby canine kidney cells. We show that both the apically targeting wild-type endotubin and a basolaterally targeted cytoplasmic domain mutant do not associate with rafts and are TX-100 soluble. The cytoplasmic tail of endotubin is sufficient for apical endosomal targeting, as chimeras with the endotubin cytoplasmic domain and Tac transmembrane and extracellular domains are efficiently targeted to the apical endosomal compartment. Furthermore, we show that overexpression of these chimeras results in their missorting to the basolateral membrane, indicating that the apical sorting process is a saturable event. These results show that cells contain machinery in both the biosynthetic and endosomal compartments that recognize cytoplasmic apical sorting signals.

Cytoarchitecture and Physical Properties of Cytoplasm: Volume, Viscosity, Diffusion, Intracellular Surface Area

Classical biochemistry is founded on several assumptions valid in dilute aqueous solutions that are often extended without question to the interior milieu of intact cells. In the first section of this chapter, we present these assumptions and briefly examine the ways in which the cell interior may depart from the conditions of an ideal solution. In the second section, we summarize experimental evidence regarding the physical properties of the cell cytoplasm and their effect on the diffusion and binding of macromolecules and vesicles. While many details remain to be worked out, it is clear that the aqueous phase of the cytoplasm is crowded rather than dilute, and that the diffusion and partitioning of macromolecules and vesicles in cytoplasm is highly restricted by steric hindrance as well as by unexpected binding interactions. Furthermore, the enzymes of several metabolic pathways are now known to be organized into structural and functional units with specific localizations in the solid phase, and as much as half the cellular protein content may also be in the solid phase.

Partition of macromolecules and cell particles in aqueous two-phase systems based on hydroxypropyl starch and poly(ethylene glycol)

The partition behavior of proteins, nucleic acids, cell membranes, cell organelles and whole cells has been studied in liquid-liquid two-phase systems composed of water, poly-(ethylene glycol), and an hydroxypropyl starch. The properties of the systems are in many respects comparable with the traditional poly(ethylene glycol)-dextran systems, but the cost is reduced to around one-fifth.

Excluded volume effects on the partition of macromolecules between two liquid phases

Partition parameters of several proteins and other macromolecules are measured in an aqueous two-phase liquid system containing polyethylene glycol and phosphate buffer. Distribution of macromolecules is a function of the relative volume excluded to the macromolecules in the two phases. A simple model with no adjustable parameters yields covolumes of the macromolecules with the polyethylene glycol. Covolumes are used to estimate effective molecular volumes and the magnitudes of excluded volume effects. The same approach is applied to mixtures of macromolecules.

Multi-stage mixer-settler planet centrifuge : Preliminary studies on partition of macromolecules with organic-aqueous and aqueous-aqueous two-phase solvent systems

A rotary-seal-free planetary centrifuge holds a separation column which consists of multiple partition units ( ca. 200) connected in series with transfer tubes. In the cavity of each partition unit the transfer tube extends to form a mixer which vibrates to stir the contents under an oscillating force field generated by the planetary motion of the centrifuge. Consequently, solutes locally introduced at the inlet of the column are subjected to an efficient partition process in each partition unit and separated according to their partition coefficients. The mixer tube equipped with a flexible silicone rubber joint was found to produce excellent results for partition with viscous polymer phase systems. The capability of the method was demonstrated on separation of cytochrome c and lysozyme using a PEG-aqueous dibasic potassium phosphate-aqueous two-phase solvent system.

Cyclohexanone/1,2‐dichloroethane as eluents in GPC of polymers

AbstractThe changes in GPC elution volumes and separation efficiency were studied for polystyrene reference materials and poly(methyl methacrylate) fractions with cyclohexanone, 1,2‐dichloroethane and their mixtures as eluents and silanized CPG‐10 porous glass as packing. Despite careful selection of the pair of the solvents interaction differences in the system gel‐eluent‐polymer caused such secondary phenomena as adsorption and/or partition of macromolecules and do not allow to determine the influence of solvent viscosity on the elution volumes of polymers in GPC.

Diffusion and partitioning of macromolecules within finely porous glass

AbstractMeasurements of diffusion and partitioning of nearly monodisperse polystyrene, having molecular weights of from 600 to 670,000, in chloro‐form and dichloroethane and of two proteins in aqueous solution were made with leached borosilicate glass cubes having a narrow pore size distribution and pore radi of from 2.5 to 47.6 nm. With increasing ratio of molecular to pore size, the partition coefficient for all solutes decreased; the ratio of effective to bulk diffusivity decreased for the proteins but remained constant for polystyrene. This suggests that polystyrene behaved like a free‐draining macromolecule under the conditions of this study.

Partition of virus particles in a liquid two-phase system

1. 1. The partition coefficients of Helix pomatia hemocyanin, tobacco mosaic virus, the bacteriophages T 3, T 2 and T 4, and vaccina virus have been determined in the phase system dextran-methylcellulose-water. 2. 2. A proportionality between the logarithm of the partition coefficient and the surface area of the particles was found, in accordance with the Brønsted theory on the partition of macromolecules in two-phase systems. 3. 3. The possibility of using polymer phase systems for the analysis and fractionation of virus suspensions is shown by a counter-current experiment with tobacco mosaic virus.