Influence of salts on electrostatic interactions between poliovirus and membrane filters.

Neither solutions of salts nor solutions of detergents or of an alcohol at pH 4 are capable of eluting poliovirus adsorbed to membrane filters. However, solutions containing both a salt, such as magnesium chloride or sodium chloride, and a detergent or alcohol at pH 4 were capable of eluting adsorbed virus. The ability of ions to promote elution of virus at low pH in the presence of detergent or alcohol was dependent on the size of the ions and the ionic strength of the medium. These results suggest that both electrostatic and hydrophobic interactions are important in maintaining virus adsorption to membrane filters. Hydrophobic interactions can be disrupted by detergents or alcohols. It appears that electrostatic interactions can be disrupted by raising the pH of a solution or by adding certain salts. Disruption of either electrostatic or hydrophobic interactions alone does not permit efficient elution of the adsorbed virus at low pHs. However, when both interactions are disrupted, most of the poliovirus adsorbed to membrane filters is eluted, even at pH 4.     

Influence of salts on electrostatic interactions between poliovirus and membrane filters

Neither solutions of salts nor solutions of detergents or of an alcohol at pH 4 are capable of eluting poliovirus adsorbed to membrane filters. However, solutions containing both a salt, such as magnesium chloride or sodium chloride, and a detergent or alcohol at pH 4 were capable of eluting adsorbed virus. The ability of ions to promote elution of virus at low pH in the presence of detergent or alcohol was dependent on the size of the ions and the ionic strength of the medium. These results suggest that both electrostatic and hydrophobic interactions are important in maintaining virus adsorption to membrane filters. Hydrophobic interactions can be disrupted by detergents or alcohols. It appears that electrostatic interactions can be disrupted by raising the pH of a solution or by adding certain salts. Disruption of either electrostatic or hydrophobic interactions alone does not permit efficient elution of the adsorbed virus at low pHs. However, when both interactions are disrupted, most of the poliovirus adsorbed to membrane filters is eluted, even at pH 4.     

Influence of salts on virus adsorption to microporous filters

We investigated the direct and indirect effects of mono-, di-, and trivalent salts (NaCl, MgCl(2), and AlCl(3)) on the adsorption of several viruses (MS2, PRD-1, phiX174, and poliovirus 1) to microporous filters at different pH values. The filters studied included Millipore HA (nitrocellulose), Filterite (fiberglass), Whatman (cellulose), and 1MDS (charged-modified fiber) filters. Each of these filters except the Whatman cellulose filters has been used in virus removal and recovery procedures. The direct effects of added salts were considered to be the effects associated with the presence of the soluble salts. The indirect effects of the added salts were considered to be (i) changes in the pH values of solutions and (ii) the formation of insoluble precipitates that could adsorb viruses and be removed by filtration. When direct effects alone were considered, the salts used in this study promoted virus adsorption, interfered with virus adsorption, or had little or no effect on virus adsorption, depending on the filter, the virus, and the salt. Although we were able to confirm previous reports that the addition of aluminum chloride to water enhances virus adsorption to microporous filters, we found that the enhanced adsorption was associated with indirect effects rather than direct effects. The increase in viral adsorption observed when aluminum chloride was added to water was related to the decrease in the pH of the water. Similar results could be obtained by adding HCl. The increased adsorption of viruses in water at pH 7 following addition of aluminum chloride was probably due to flocculation of aluminum, since removal of flocs by filtration greatly reduced the enhancement observed. The only direct effect of aluminum chloride on virus adsorption that we observed was interference with adsorption to microporous filters. Under conditions under which hydrophobic interactions were minimal, aluminum chloride interfered with virus adsorption to Millipore, Filterite, and 1MDS filters. In most cases, less than 10% of the viruses adsorbed to filters in the presence of a multivalent salt and a compound that interfered with hydrophobic interactions (0.1% Tween 80 or 4 M urea).     

Influence of Salts on Virus Adsorption to Microporous Filters

We investigated the direct and indirect effects of mono-, di-, and trivalent salts (NaCl, MgCl₂, and AlCl₃) on the adsorption of several viruses (MS2, PRD-1, X174, and poliovirus 1) to microporous filters at different pH values. The filters studied included Millipore HA (nitrocellulose), Filterite (fiberglass), Whatman (cellulose), and 1MDS (charged-modified fiber) filters. Each of these filters except the Whatman cellulose filters has been used in virus removal and recovery procedures. The direct effects of added salts were considered to be the effects associated with the presence of the soluble salts. The indirect effects of the added salts were considered to be (i) changes in the pH values of solutions and (ii) the formation of insoluble precipitates that could adsorb viruses and be removed by filtration. When direct effects alone were considered, the salts used in this study promoted virus adsorption, interfered with virus adsorption, or had little or no effect on virus adsorption, depending on the filter, the virus, and the salt. Although we were able to confirm previous reports that the addition of aluminum chloride to water enhances virus adsorption to microporous filters, we found that the enhanced adsorption was associated with indirect effects rather than direct effects. The increase in viral adsorption observed when aluminum chloride was added to water was related to the decrease in the pH of the water. Similar results could be obtained by adding HCl. The increased adsorption of viruses in water at pH 7 following addition of aluminum chloride was probably due to flocculation of aluminum, since removal of flocs by filtration greatly reduced the enhancement observed. The only direct effect of aluminum chloride on virus adsorption that we observed was interference with adsorption to microporous filters. Under conditions under which hydrophobic interactions were minimal, aluminum chloride interfered with virus adsorption to Millipore, Filterite, and 1MDS filters. In most cases, less than 10% of the viruses adsorbed to filters in the presence of a multivalent salt and a compound that interfered with hydrophobic interactions (0.1% Tween 80 or 4 M urea).     

Protein stabilization and destabilization by guanidinium salts

Preferential interactions of bovine serum albumin were measured with guanidine sulfate, guanidine acetate, and guanidine hydrochloride. The results showed an increasing preferential hydration with increasing salt concentration for the sulfate, positive preferential salt binding for the hydrochloride, and an intermediate situation for the acetate. These results correlate well with the known effects of the three salts on protein stability, namely, the stabilizing effect of guanidine sulfate and the denaturing effect of guanidine hydrochloride. Comparison of guanidinium and magnesium salts indicated that the substitution of guanidinium ion for Mg2+ decreases the preferential hydration and increases the preferential salt binding, suggesting that the perturbation by guanidinium ion binding of the surface free energy is greater than that by Mg2+ ion. It was concluded that guanidine salts are not a special class, but their activity toward proteins is modulated by the same fine balance between hydration and salt binding to protein as in the case of other salts, with the second factor being stronger in guanidine salts.     

Control of antibody high and low molecular weight species by depth filtration-based cell culture harvesting

Depth filtration-based harvesting is widely used in mAb manufacturing to remove cell and process-related impurities. However, it has not been studied on control of product-related impurities, which are very critical for product quality. In this article, we studied the interactions of depth filter with high and low molecular weight species (HMWs and LMWs) for their direct removal from cell culture. The process parameters (filter, loading, temperature, and flux) were evaluated for adsorption of HMWs and LMWs by depth filters. The adsorption is significantly dependent on filter media and loading capacity and is mainly on the basis of hydrophobic interaction during harvesting. The HMW and LMW species were characterized as HMW1, HMW2, LMW1, and LMW2. The increasing binding from LMW2 to LMW1, HMW1, and HMW2 is correlated with their increasing hydrophobicity score. Adsorption using enriched HMW sample demonstrated similar total protein binding capacity (36–40 g/m²) between depth filters D0HC and X0HC. However, X0HC has stronger HMW binding than D0HC (71% vs 43% of bound protein), indicating more hydrophobic interaction in X0HC. HMW2 DBC on X0HC reached 12 g/m², similar to protein binding on hydrophobic interaction membrane adsorbers. Further study showed LMW can induce HMW formation. This study provides a critical understanding of HMW and LMW interaction with depth filters. The strategy of HMW and LMW control by depth filtration-based harvesting was implemented successfully in mAb manufacturing.     

Hydrophobicity and adhesion to fish cells and mucus of Vibrio strains isolated from infected fish

The hydrophobicity of 44 Vibrio strains isolated from cultured, diseased gilt-head sea bream (Sparus aurata) was determined. Three different methods were used: (1) microbial adhesion to hydrocarbons (MATH), either with phosphate buffer or with phosphate urea magnesium sulfate (PUM) buffer, (2) aggregation in the presence of salt solutions (SAT), and (3) adhesion to nitrocellulose filters (NCF). The results show that experimental conditions exerted a significant influence on hydrophobicity. Thus, Kendall rank coefficients showed the presence of correlation only for SAT and NCF, and for SAT and the MATH assay with PUM buffer. Moreover, no relationships were observed between the bacterial hydrophobicity estimated with the methods mentioned above and the ability of the strains to adhere to fish mucus or cells. These results indicate that adhesion of pathogenic Vibrio strains to host surfaces is mediated mainly by specific receptor interactions, instead of by hydrophobic interactions.     

Use of modified diatomaceous earth for removal and recovery of viruses in water.

Diatomaceous earth was modified by in situ precipitation of metallic hydroxides. Modification decreased the negative charge on the diatomaceous earth and increased its ability to adsorb viruses in water. Electrostatic interactions were more important than hydrophobic interactions in virus adsorption to modified diatomaceous earth. Filters containing diatomaceous earth modified by in situ precipitation of a combination of ferric chloride and aluminum chloride adsorbed greater than 80% of enteroviruses (poliovirus 1, echovirus 5, and coxsackievirus B5) and coliphage MS2 present in tap water at ambient pH (7.8 to 8.3), even after filtration of 100 liters of tap water. Viruses adsorbed to the filters could be recovered by mixing the modified diatomaceous earth with 3% beef extract plus 1 M NaCl (pH 9).     

Use of modified diatomaceous earth for removal and recovery of viruses in water.

Diatomaceous earth was modified by in situ precipitation of metallic hydroxides. Modification decreased the negative charge on the diatomaceous earth and increased its ability to adsorb viruses in water. Electrostatic interactions were more important than hydrophobic interactions in virus adsorption to modified diatomaceous earth. Filters containing diatomaceous earth modified by in situ precipitation of a combination of ferric chloride and aluminum chloride adsorbed greater than 80% of enteroviruses (poliovirus 1, echovirus 5, and coxsackievirus B5) and coliphage MS2 present in tap water at ambient pH (7.8 to 8.3), even after filtration of 100 liters of tap water. Viruses adsorbed to the filters could be recovered by mixing the modified diatomaceous earth with 3% beef extract plus 1 M NaCl (pH 9).     

Characterization of virus adsorption by using DEAE-sepharose and octyl-sepharoser

Viruses were characterized by their adsorption to DEAE-Sepharose or by their elution from octyl-Sepharose by using buffered solutions of sodium chloride with different ionic strengths. Viruses whose adsorption to DEAE-Sepharose was reduced most rapidly by an increase in the sodium chloride concentration were considered to have the weakest electrostatic interactions with the solids; these viruses included MS2, E1, and phiX174. Viruses whose adsorption to DEAE-Sepharose was reduced least rapidly were considered to have the strongest electrostatic interactions with the column; these viruses included P1, T4, T2, and E5. All of the viruses studied adsorbed to octyl-Sepharose in the presence of 4 M NaCl. Viruses that were eluted most rapidly following a decrease in the concentration of NaCl were considered to have the weakest hydrophobic interactions with the column; these viruses included phiX174, CB4, and E1. Viruses that were eluted least rapidly from the columns after the NaCl concentration was decreased were considered to have the strongest hydrophobic interactions with the column; these viruses included f2, MS2, and E5.     

Efficient Filtration and Sizing of Viruses with Membrane Filters

Untreated membrane filters retain viruses by adsorption, as well as by physical restriction which occurs when the pore diameter of the filter is smaller than that of the virus particle. As originally recommended by Elford, membranes had to be pretreated with proteinaceous material to preclude virus adsorption. However, coating materials that prevent adsorption of certain viruses do not necessarily prevent adsorption of other viruses. In contrast to proteins, salts enhance virus adsorption. Viruses treated with sodium lauryl sulfate to reduce the surface tension, or purified viruses in distilled water, are not adsorbed to membranes. A procedure is recommended by which viruses may be passed through membranes with a porosity twice the diameter of the virus. Such filtrates, which contain 50 to 100% of the initial virus concentration, should be used for sizing viruses by subsequent filtration through smaller pores. The determination of virus size would then be based on the major population of particles in the virus suspension. In the past, as little as 0.1 to 0.001% of the initial virus population was the basis for size determination, because more than 99.9% of the virus was often lost by adsorption to membranes during the clarifying procedures.     

Cholesterol promotes the interaction of Alzheimer β-amyloid monomer with lipid bilayer

Cholesterol in the plasma membrane plays an important role in the pathogenesis of Alzheimer's disease, but the exact function of cholesterol in the regulation of amyloid-β (Aβ) generation, aggregation, and toxicity remains elusive. To gain insight into the bioactivity of cholesterol, we investigate the effect of cholesterol levels on the interaction of Aβ(1-42) monomer with the zwitterionic 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) bilayer containing different mole fractions of cholesterol from χ=0, 0.2, to 0.4 using all-atom molecular dynamics simulations. Simulation results show that an increased cholesterol level alters the structure, dynamics, and surface chemistry of the lipid bilayer, leading to increased bilayer thickness, hydrophobic chain order, surface hydrophobicity, and decreased lipid mobility. All these effects significantly promote the binding of Aβ to the lipid bilayer. Mechanistically, the adsorption of Aβ on the bilayer is a cooperative process. First, charged residues act as anchors to establish the initial binding of Aβ to phosphate headgroups of the bilayer driven by electrostatic interactions, which further facilitates hydrophobic residues to reside on the bilayer. Once hydrophobic residues especially from C-terminus are locked on the bilayer, the interactions among charged residues, lipid bilayer, and calcium ions are optimized to provide additional attractive forces to stabilize Aβ adsorbed on or inserted into the lipid bilayer. Inclusion of cholesterol makes this binding process more energetically favorable. Upon adsorption on the bilayer, Aβ appears to preferentially adopt α-helical or unstructured conformation. This work supports that cholesterol acts as a promoter for Aβ--membrane interactions, which would facilitate Aβ aggregation and membrane insertion.     

Stimulatory effect of ammonium sulfate at high concentrations on the aminoacylation of tRNA and tRNA-like molecules

The influence of various salts on the aminoacylation of tRNA(Val) and the tRNA-like structure from turnip yellow mosaic virus RNA by yeast valyl-tRNA synthetase has been studied. As expected, increasing the concentration of salts inhibits the enzymatic reaction. However, in the presence of high concentration of ammonium sulfate, and only this salt, the inhibitory effect is suppressed. Under such conditions, the aminoacylation becomes comparable to that measured in the absence of salt. It was shown that ammonium sulfate affects both the catalytic rate of the reaction and the affinity between valyl-tRNA synthetase and the RNAs. Because the affinity between the partners in the complex is increased when the concentration of the salt is high, it is suggested that hydrophobic effects are involved in tRNA/synthetase interactions.     

Partition of isomeric dipeptides in poly(ethylene glycol)/magnesium sulfate aqueous two-phase systems

A series of isomeric dipeptides, i.e., those containing identical residues but in different order such as Trp-Gly versus Gly-Trp, was partitioned in a poly(ethylene glycol) (PEG)/magnesium sulfate (MgSO4) aqueous two-phase system. Dipeptides having a more hydrophobic character favored the upper (PEG) phase. Moreover, the partition coefficients for isomeric dipeptides are different, with the partition coefficients for dipeptides containing the more hydrophobic residue in the C-terminal position being, in general, greater than the partition coefficients for corresponding isomers which contain the more hydrophobic residue in the N-terminal position. These observations can be attributed to the different interactions that the isomers have with specific two-phase systems.     

Plasmid DNA purification by selective calcium silicate adsorption of closely related impurities

The selective adsorption of supercoiled plasmid, open-circular plasmid, and genomic DNA to gyrolite, a compound from the class of crystalline calcium silicate hydrates, is investigated and exploited for purification purposes. Genomic DNA and open-circular plasmid bind to gyrolite adsorbents with greater affinity than the more conformationally constrained supercoiled plasmid. As such, the gyrolite adsorbents are an economical and scaleable alternative to chromatographic purification for the removal of DNA impurities from solutions containing supercoiled plasmid. The advantage of gyrolite adsorbents is their lower unit price and ability to selectively adsorb DNA impurities without binding supercoiled plasmid under certain conditions. The effects of ionic strength, temperature, chelating agent, divalent cation, and lyotropic salts on adsorption of highly purified plasmid are studied to understand the forces that bind DNA to gyrolite, a structure with hydrophilic and hydrophobic characteristics. The results indicate that DNA binding is governed by hydrogen bonding, electrostatic bridging with divalent cations, shielding of electrostatic repulsion, hydrophobic adsorption, and disruption of integral surface water layer on gyrolite. On the basis of results from a range of Hofmeister series salts, strongly hydrated anions may enhance DNA adsorption by promoting hydrophobic interactions between DNA and gyrolite. Conversely, the very weakly hydrated chaotrope I(-) may enhance adsorption by strongly associating with hydrophobic siloxanes of gyrolite, thereby disrupting an integral water layer, which competes for hydrogen bonding sites.     

Adsorptive immobilization of erythrocyte membrane

Immobilization of human erythrocyte membrane was carried out by adsorption on Fractosil, a porous form of silica. Acetylcholinesterase (AChE) was chosen as a representative membrane enzyme in this study. Dependency of adsorption on membrane concentration was determined. Positive cooperative interactions that occurred in the process of immobilization increased stability. Presence of hydrophobic ligands on derivatized Fractosil was found to enhance stability of immobilized preparations making them more effective for use in continuous catalytic transformations. It is suggested that adsorptive immobilization of membrane structures such as the human erythrocyte membrane fragments on Fractosil and other inexpensive supports may provide a convenient procedure for utilization of their catalytic potential. Such preparations may be used in diagnostic kits or for construction of biosensors.     

Characterization of Virus Adsorption by Using DEAE-Sepharose and Octyl-Sepharose†

Viruses were characterized by their adsorption to DEAE-Sepharose or by their elution from octyl-Sepharose by using buffered solutions of sodium chloride with different ionic strengths. Viruses whose adsorption to DEAE-Sepharose was reduced most rapidly by an increase in the sodium chloride concentration were considered to have the weakest electrostatic interactions with the solids; these viruses included MS2, E1, and X174. Viruses whose adsorption to DEAE-Sepharose was reduced least rapidly were considered to have the strongest electrostatic interactions with the column; these viruses included P1, T4, T2, and E5. All of the viruses studied adsorbed to octyl-Sepharose in the presence of 4 M NaCl. Viruses that were eluted most rapidly following a decrease in the concentration of NaCl were considered to have the weakest hydrophobic interactions with the column; these viruses included X174, CB4, and E1. Viruses that were eluted least rapidly from the columns after the NaCl concentration was decreased were considered to have the strongest hydrophobic interactions with the column; these viruses included f2, MS2, and E5.     

Covalent chromatography and salt-promoted thiophilic adsorption

Covalent chromatography on 3-(2-pyridyl disulfido)-2-hydroxypropyl agarose, abbreviated PyS2, turns out to involve more complex interactions than has been supposed heretofore. Unexpectedly, the sorption is highly salt dependent. The relative affinities for serum proteins have therefore been determined in the absence and presence of different types of salts at different salt concentrations and with different degrees of ligand substitution on the adsorbent. In the presence of water-structuring salts the PyS2-gel shows an adsorption pattern for serum proteins resembling that of the "thiophilic" T-gel (J. Porath; F. Maisano, and M. Belew (1985) FEBS Lett. 185, 306-310). Superimposed on thiophilic adsorption we have found, as expected, covalent attachment of thiol-containing proteins. Also the thiol-disulfide exchange increases from 4-5% in the absence of potassium sulfate or sodium chloride up to about 40% of the applied serum proteins when such a water-structuring salt is present. We have thus shown that the interaction of a protein with the ligand is greatly facilitated by a water-structuring salt--and in this case the product is a covalently as well as a thiophilically immobilized protein. A cautious interpretation of protein interaction phenomena is justified whenever ligands containing sulfide, disulfide, or pi-electron-rich structures such as aromatic moieties are involved.     

Structural basis for bile salt inhibition of pancreatic phospholipase A2

Bile salt interactions with phospholipid monolayers of fat emulsions are known to regulate the actions of gastrointestinal lipolytic enzymes in order to control the uptake of dietary fat. Specifically, on the lipid/aqueous interface of fat emulsions, the anionic portions of amphipathic bile salts have been thought to interact with and activate the enzyme group-IB phospholipase A2 (PLA2) derived from the pancreas. To explore this regulatory process, we have determined the crystal structures of the complexes of pancreatic PLA2 with the naturally occurring bile salts: cholate, glycocholate, taurocholate, glycochenodeoxycholate, and taurochenodeoxycholate. The five PLA2-bile salt complexes each result in a partly occluded active site, and the resulting ligand binding displays specific hydrogen bonding interactions and extensive hydrophobic packing. The amphipathic bile salts are bound to PLA2 with their polar hydroxyl and sulfate/carboxy groups oriented away from the enzyme's hydrophobic core. The impaired catalytic and interface binding functions implied by these structures provide a basis for the previous numerous observations of a biphasic dependence of the rate of PLA2 catalyzed hydrolysis of zwitterionic glycerophospholipids in the presence of bile salts. The rising or activation phase is consistent with enhanced binding and activation of the bound PLA2 by the bile salt induced anionic charge in a zwitterionic interface. The falling or inhibitory phase can be explained by the formation of a catalytically inert stoichiometric complex between PLA2 and any bile salts in which it forms a stable complex. The model provides new insight into the regulatory role that specific PLA2-bile salt interactions are likely to play in fat metabolism.     

Destruction of the outer membrane permeability barrier of Escherichia coli by heat treatment

Heat treatment of a wild-type Escherichia coli strain at 55 degrees C in 50 mM Tris-hydrochloride buffer with or without 10 mM magnesium sulfate or HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) buffer at pH 8.0 caused an increase in cell surface hydrophobicity. By determining the location of n-hexadecane droplets attached to cells by phase-contrast microscopy, the septal and polar regions of heated cells appeared to become the most frequently hydrophobic. Some of the lipopolysaccharide molecules in the outer membrane were released from heated cells, and the cells became susceptible to the hydrolytic action of added phospholipase C. Heat-treated cells also became permeable to the hydrophobic dye crystal violet, which was added externally. The release of part of the outer membrane by heat treatment appeared to bring about the disorganization of the outer membrane structure and, as a consequence, to result in the partial disruption of the permeability barrier function of the outer membrane. Tris was found to enhance damage to the outer membrane by heat.