Adsorption of viruses to charge-modified silica

The purpose of this study was to provide a clearer understanding of virus adsorption, focusing specifically on the role of electrostatic interactions between virus particles and adsorbent surfaces. The adsorption of poliovirus 1, reovirus types 1 and 3, and coliphages MS-2 and T2 to colloidal silica synthetically modified to carry either positive or negative surface charge was evaluated. Adsorption experiments were performed by combining virus and silica in 0.1-ionic-strength buffers of pH 4.0, 6.4, and 8.5. Samples agitated for specified adsorption periods were centrifuged to pellet adsorbent particles plus adsorbed virus, and the supernatants were assayed for unadsorbed virus. All viruses adsorbed exclusively to negatively charged silica at pH values below their isoelectric points, i.e., under conditions favoring a positive surface charge on the virions. Conversely, all viruses adsorbed exclusively to positively charged silica at pH values above their isoelectric points, i.e., where virus surface charge is negative. Viruses in near-isoelectric state adsorbed to all types of silica, albeit to a lesser degree.     

Influence of pH and electrolyte composition on adsorption of poliovirus by soils and minerals.

The pH and the nature an concentration of simple electrolytes influenced the interaction of poliovirus type 2 with three soils, a sand, and a clay mineral. In electrolytes above pH 9 the virus was not adsorbed extensively to the substrates, but below pH 7 almost all virus was bound. For each adsorbent there was a characteristic pH region of transition from strong to weak uptake. Differences between the soils in virus uptake were shown to parallel their pH-dependent mineral. In electrolytes above pH 9 the virus was not adsorbed extensively to the substrates, but below pH 7 almost all virus was bound. For each adsorbent there was a characteristic pH region of transition from strong to weak uptake. Differences between the soils in virus uptake were shown to parallel their pH-dependent mineral. In electrolytes above pH 9 the virus was not adsorbed extensively to the substrates, but below pH 7 almost all virus was bound. For each adsorbent there was a characteristic pH region of transition from strong to weak uptake. Differences between the soils in virus uptake were shown to parallel their pH-dependent charge properties, as determined by whole-particle microelectrophoresis. Only when the pH was close to or above the critical region was uptake increased with electrolyte concentration. The transition region for all substrates was above pH 7.5 the isoelectric point of the virus. Thus, it appears that when both the virus and substrate are highly negative charged, repulsive electrostatic effects may exceed inherent attractive interactions, thereby inhibiting adsorption.     

Influence of pH and electrolyte composition on adsorption of poliovirus by soils and minerals

The pH and the nature an concentration of simple electrolytes influenced the interaction of poliovirus type 2 with three soils, a sand, and a clay mineral. In electrolytes above pH 9 the virus was not adsorbed extensively to the substrates, but below pH 7 almost all virus was bound. For each adsorbent there was a characteristic pH region of transition from strong to weak uptake. Differences between the soils in virus uptake were shown to parallel their pH-dependent mineral. In electrolytes above pH 9 the virus was not adsorbed extensively to the substrates, but below pH 7 almost all virus was bound. For each adsorbent there was a characteristic pH region of transition from strong to weak uptake. Differences between the soils in virus uptake were shown to parallel their pH-dependent mineral. In electrolytes above pH 9 the virus was not adsorbed extensively to the substrates, but below pH 7 almost all virus was bound. For each adsorbent there was a characteristic pH region of transition from strong to weak uptake. Differences between the soils in virus uptake were shown to parallel their pH-dependent charge properties, as determined by whole-particle microelectrophoresis. Only when the pH was close to or above the critical region was uptake increased with electrolyte concentration. The transition region for all substrates was above pH 7.5 the isoelectric point of the virus. Thus, it appears that when both the virus and substrate are highly negative charged, repulsive electrostatic effects may exceed inherent attractive interactions, thereby inhibiting adsorption.     

Simple method for the concentration of influenza virus from allantoic fluid on microporous filters.

Membrane filter adsorption-elution is an efficient method for concentration and partial purification of several types of viruses from various aqueous solutions. For efficient virus adsorption to negatively charged filters, the sample is adjusted to pH 3.5 and trivalent salts are added before filtration. Since influenza virus is sensitive to extremes in pH, it cannot be concentrated by ordinary filters. Zeta Plus filters, which have a net positive charge of up to 5 or 6, were evaluated for the concentration of influenza virus from infectious allantoic fluids. Influenza virus efficiently adsorbed to Zeta Plus filters at pH 6, and addition of salts was not necessary. Adsorbed virus was eluted in a small volume of 2% bovine serum albumin plus 1 M NaCl at pH 10. By this procedure, viruses in 100 ml of allantoic fluid were concentrated to a final volume of 8 ml, with an average recovery efficiency of 71.0%.     

原位椭圆偏振术研究牛血清清蛋白在固/液界面的吸附

用原位椭圆偏振术系统研究了硅片表面因素及缓冲液环境因素对牛血清清蛋白在固/液界面吸附的影响。在生理条件下,疏水表面与亲水表面相比BSA吸附量较大。随着硅片表面电荷密度增加,BSA吸附量增加。BSA吸附量当体溶液pH值等于BSA等电点时达到最大。而随着体溶液离子强度增加,BSA吸附量亦上升。实验结果提示:除了熵驱动作用之外,硅片表面与BSA分子及BSA分子之间的静电作用在BSA吸附中起着十分重要的作用。     

Adsorption of reovirus by minerals and soils.

Adsorption of [35S]methionine-labeled reovirus by 30 dry soils, minerals, and finely ground rocks suspended in synthetic freshwater at pH 7 was investigated to determine the conditions necessary for optimum virus removal during land application of wastewaters. All of the minerals and soils studied were excellent adsorbents of reovirus, with greater than 99% of the virus adsorbed after 1 h at 4 degrees C. Thereafter, virus remaining in suspension was significantly inactivated, and within 24 h a three to five log10 reduction in titer occurred. The presence of divalent cations, i.e., Ca2+ and Mg2+, in synthetic freshwater enhanced removal, whereas soluble organic matter decreased the amount of virus adsorbed in secondary effluent. The amount of virus adsorbed by these substrates was inversely correlated with the amount of organic matter, capacity to adsorb cationic polyelectrolyte, and electrophoretic mobility. Adsorption increased with increasing available surface area, as suspended infectivity was reduced further by the more finely divided substrates. However, the organic content of the soils reduced the level of infectious virus adsorbed below that expected from surface area measurements alone. The inverse correlation between virus adsorption and substrate capacity for cationic polyelectrolyte indicates that the adsorption of infectious reovirus particles is predominately a charged colloidal particle-charged surface interaction. Thus, adsorption of polyelectrolyte may be useful in predicting the fate of viruses during land application of sewage effluents and sludges.     

Adsorption of gamma-globulin, a model protein for antibody, on colloidal particles

The effect of surface properties on the adsorption of bovine gamma-globulin, a model protein for antibody, was studied. Polystyrene latex (PS), hydrophilic copolymer lattices of styrene/2-hydroxyethyl methacrylate [P(S/HEMA)], styrene/ methacrylic acid [P(S/MAA)] and methyl methacrylate/ 2-hydroxyethyl methacrylate [P(MMA/HEMA)], and colloidal silica were used. The adsorption isotherms of gamma-globulin on these colloidal particles were measured as a function of pH and ionic strength. The hydrophilic particles showed low affinities for gamma-globulin at alkaline pH, while PS showed high affinities for gamma-globulin over the whole range of pH and ionic strength. The gamma-globulin adsorption on hydrophilic particles was highly reversible with respect to the pH and ionic strength compared with that on PS. These differences indicate that the dominant driving forces of adsorption are related to the hydrophilicity of particles. The adsorption isotherms of all colloidal particles showed the plateau values, and the order of maximum values of plateau adsorption was P(S/MAA) > PS or P(S/HEMA), silica > P(MMA/HEMA). Thus, they were also affected by the charged groups and the hydrophilicity of the surfaces. On the other hand, the plateau values of all colloidal particles were more or less symmetrical with a maximum at around the isoelectric point of gamma-globulin at an ionic strength of 0.01. This behavior is attributed to the important role of the lateral interaction between the adsorbed molecules at low ionic strength.     

Viral aggregation: buffer effects in the aggregation of poliovirus and reovirus at low and high pH.

The effects of the buffer employed in maintaining a given pH value were tested on the aggregation of two viruses, poliovirus and reovirus. Poliovirus was found to aggregate at pH values of 6 and below, but not at pH 7 or above, except in borate buffer. Reovirus aggregated at pH 4 and below, but was found to aggregate only in acetate or tris(hydroxymethyl)aminomethane-citrate buffers at pH 5. Other buffers tested for aggregation of reovirus at pH 5 (succinate, citrate, and phosphate-citrate) induced little aggregation. No significant aggregation was found for reovirus at pH 6 and above. For both viruses, the most effective aggregation was induced by buffers having a substantial monovalently charged anionic component, such as acetate at pH 5 and 6 or citrate at pH 3. Cationic buffers at low pH, such as glycine, were generally weaker in aggregating ability than anionic buffers at the same pH. These results, when correlated with the isoelectric point of the viruses (poliovirus at pH 8.2; reovirus at pH 3.9) indicated that both viruses aggregated strongly when their overall charge was positive, but only under certain circumstances when their overall charge was negative. Although reovirus aggregated massively at its isoelectric point, poliovirus remained dispersed at its isoelectric point. The conclusion can be drawn that those pH and buffer conditions which induced aggregation of one virus do not necessarily induce it in another.     

Viral aggregation: buffer effects in the aggregation of poliovirus and reovirus at low and high pH.

The effects of the buffer employed in maintaining a given pH value were tested on the aggregation of two viruses, poliovirus and reovirus. Poliovirus was found to aggregate at pH values of 6 and below, but not at pH 7 or above, except in borate buffer. Reovirus aggregated at pH 4 and below, but was found to aggregate only in acetate or tris(hydroxymethyl)aminomethane-citrate buffers at pH 5. Other buffers tested for aggregation of reovirus at pH 5 (succinate, citrate, and phosphate-citrate) induced little aggregation. No significant aggregation was found for reovirus at pH 6 and above. For both viruses, the most effective aggregation was induced by buffers having a substantial monovalently charged anionic component, such as acetate at pH 5 and 6 or citrate at pH 3. Cationic buffers at low pH, such as glycine, were generally weaker in aggregating ability than anionic buffers at the same pH. These results, when correlated with the isoelectric point of the viruses (poliovirus at pH 8.2; reovirus at pH 3.9) indicated that both viruses aggregated strongly when their overall charge was positive, but only under certain circumstances when their overall charge was negative. Although reovirus aggregated massively at its isoelectric point, poliovirus remained dispersed at its isoelectric point. The conclusion can be drawn that those pH and buffer conditions which induced aggregation of one virus do not necessarily induce it in another.     

Kinetic and circular dichroism studies of enzymes adsorbed on ultrafine silica particles

Negatively charged ultrafine silica particles (average diameter 20 nm) were used as support materials for adsorption immobilization of porcine trypsin, horseradish peroxidase, and bovine catalase under various conditions, and the changes in the enzyme activities and the circular dichroism (CD) spectra of these enzymes upon adsorption were measured. Since the light scattering intensity of the ultrafine particles was very low, the activities and the CD spectra of the enzymes adsorbed on the particle surfaces could be measured. The enzymes adsorbed at pH around and above their isoelectric points (pI) showed high activities. On the other hand, the enzymes adsorbed at pHs below their pI had significantly diminished activities and showed large CD spectral changes upon adsorption. The extent of CD spectral changes in the enzymes upon adsorption correlated very closely with that of the activity reduction. Therefore, the conformational changes in enzymes upon adsorption are one of the important factors that reduce the activities of adsorbed enzymes. These results demonstrate that the ultrafine particles are not only a novel support for enzyme immobilization but also are helpful for the molecular understanding of the immobilized enzymes. Correspondence to: A. Kondo     

Electrophoretic mobilities of RNA tumor viruses. Studies by Doppler-shifted light scattering spectroscopy.

We have used laser beat frequency light scattering spectroscopy to measure, at several pH values, the electrophoretic mobilities of purified avian myeloblastosis (AMV), murine leukemia (MuLV), murine mammary tumor (MuMTV), and feline leukemia (FeLV) viruses. The mobilities of these viruses are similar at pH greater than or equal to7 (-2.7 to -3.2 X 10(-4) (cm/sec)/(V/cm). The isoelectric points of MuLV and AMV are apparently less than pH 3, whereas for FeLV the data could be interpreted to indicate an isoelectric point between 3 and 5. Using a Debye-Hückel model to describe the interaction between electrolytes and virus, we show that our values for the mobility of MuMTV, obtained in ionic strength 0.005, are consistent with the values of Sarkar et al. ((1973), Cancer Res. 33, 2283), obtained in ionic strength of 0.10. This model is then used to calculate surface charge densities. In terms of the density of charged groups, the RNA tumor virus envelope is not very different from the erythrocyte membrane.     

THE ORIGIN OF THE ELECTRICAL CHARGES OF COLLOIDAL PARTICLES AND OF LIVING TISSUES

1. When a solution of a salt of gelatin or crystalline egg albumin is separated by a collodion membrane from a watery solution (free from protein) a potential difference is set up across the membrane in which the protein is positively charged in the case of protein-acid salts and in which the protein is negatively charged in the case of metal proteinates. The turning point is the isoelectric point of the protein. 2. Measurements of the pH of the (inside) protein solution and of the outside watery solution show that when equilibrium is established the value pH inside minus pH outside is positive in the case of protein-acid salts and negative in the case of metal proteinates. This is to be expected when the P.D. is caused by the establishment of a Donnan equilibrium, since in that case the pH should be lower outside than inside in the case of a protein-acid salt and should be higher outside than inside in the case of a metal proteinate. 3. At the isoelectric point where the electrical charge is zero the value of pH inside minus pH outside becomes also zero. 4. It is shown that a P.D. is established between suspended particles of powdered gelatin and the surrounding watery solution and that the sign of charge of the particles is positive when they contain gelatin-acid salts, while it is negative when the powdered particles contain metal gelatinate. At the isoelectric point the charge is zero. 5. Measurements of the pH inside the powdered particles and of the pH in the outside watery solution show that when equilibrium is established the value pH inside minus pH outside is positive when the powdered particles contain a gelatin-acid salt, while the value pH inside minus pH outside is negative when the powdered particles contain Na gelatinate. At the isoelectric point the value pH inside minus pH outside is zero. 6. The addition of neutral salts depresses the electrical charge of the powdered particles of protein-acid salts. It is shown that the addition of salts to a suspension of powdered particles of gelatin chloride also diminishes the value of pH inside minus pH outside. 7. The agreement between the values 58 (pH inside minus pH outside) and the P. D. observed by the Compton electrometer is not only qualitative but quantitative. This proves that the difference in the concentration of acid (or alkali, as the case may be) in the two phases is the only cause for the observed P.D. 8. The Donnan theory demands that the P.D. of a gelatin chloride solution should be 1½ times as great as the P.D. of a gelatin sulfate solution of the same pH and the same concentration (1 per cent) of originally isoelectric gelatin. This is found to be correct and it is also shown that the values of pH inside minus pH outside for the two solutions possess the ratio of 3:2. 9. All these measurements prove that the electrical charges of suspended particles of protein are determined exclusively by the Donnan equilibrium.     

Use of amphiphilic spin labels and whole cell isoelectric focusing to assay charge characteristics of sperm surfaces.

The charge characteristics of the surface of bull and rabbit sperm were analyzed using surface-directed spin labels and whole cell isoelectric focusing. Spin label experiments tested charges believed to be localized at the membrane phospholipid-water interface. Charge properties of the glycoprotein calyx were analyzed with isoelectric focusing. Addition of charged detergents altered spin label spectra without changing the isoelectric focusing pH value. Sperm presumed to differ in the amount of adsorbed protein had different isoelectric focusing pH values, but similar spin label spectra. We conclude that these techniques are capable of monitoring charge domains on the sperm surface: one at the polar surface of the phospholipid membrane and one at the interface between the glycocalyx and the suspending fluid. Furthermore, changes in charge density are induced in unique zones of the cell surface during sperm maturation.     

Electrophoresis of absorbed RNA

The electrophoretic mobilities of adsorbed yeast ribonucleic acid have been measured as functions of pH, ionic strength, and biopolymer concentration and the results so obtained have been critically compared with those for adsorbed DNA. Like DNA, ribonucleic acid has also been found to reverse the positive charge of alumina owing to its adsorption on the solid-liquid interface. The mobilities of adsorbed RNA have been found to be less than those of adsorbed DNA under identical conditions. The observed mobilities of adsorbed heat- and alkali-denatured RNA are significantly less than those of adsorbed native RNA at a given pH and ionic strength of the medium. The electrophoretic mobilities as observed also show the evidence of RNA adsorption on the negatively charged surface of Dowex-50 resin, but practically no adsorption of RNA on the negatively charged glass surface has been predicted.     

Electrophoresis of adsorbed DNA

The electrophoresis mobilities of native calf thymus DNA adsorbed on the charged solid particles were measured by a micro-electrophoretic method as functions of pII, ionic strength, and DNA concentration. The mobility data confirm the adsorption of DNA both on the positively charged alumina and negatively charged resin particles at wide range of pH and ionic strength. The mobility data also indicate significant DNA adsorption by negatively charged glass in the acidic range of pH. The electrophoretic mobilities of DNA adsorbed on different substrate particles under identical conditions do not differ widely, indicating the major role of the adsorbed DNA rather than the covered substrate in controlling the charge behavior of the particle. The mobilities of the adsorbed DNA at salt pH are of a comparable order of magnitude to those for the dissolved DNA in solution. The mobility of the adsorbed heat-denatured and alkali-denatured DNA is lower than that of the native adsorbed DNA under identical conditions of pH and ionic strength.     

Protein adsorption orientation in the light of fluorescent probes: mapping of the interaction between site-directly labeled human carbonic anhydrase II and silica nanoparticles

Little is known about the direction and specificity of protein adsorption to solid surfaces, a knowledge that is of great importance in many biotechnological applications. To resolve the direction in which a protein with known structure and surface potentials binds to negatively charged silica nanoparticles, fluorescent probes were attached to different areas on the surface of the protein human carbonic anhydrase II. By this approach it was clearly demonstrated that the adsorption of the native protein is specific to limited regions at the surface of the N-terminal domain of the protein. Furthermore, the adsorption direction is strongly pH-dependent. At pH 6.3, a histidine-rich area around position 10 is the dominating adsorption region. At higher pH values, when the histidines in this area are deprotonated, the protein is also adsorbed by a region close to position 37, which contains several lysines and arginines. Clearly the adsorption is directed by positively charged areas on the protein surface toward the negatively charged silica surface at conditions when specific binding occurs.     

Concentration of poliovirus from tap water using positively charged microporous filters.

Microporous filters that are more electropositive than the negatively charged filters currently used for virus concentrations from water by filter adsorption-elution methods were evaluated for poliovirus recovery from tap water. Zeta Plus filters composed of diatomaceous earth-cellulose-"charge-modified" resin mixtures and having a net positive charge of up to pH 5 to 6 efficiently adsorbed poliovirus from tap water at ambient pH levels 7.0 to 7.5 without added multivalent cation salts. The adsorbed virus were eluted with glycine-NaOH, pH 9.5 to 11.5. Electropositive asbestos-cellulose filters efficiently adsorbed poliovirus from tap water without added multivalent cation salts between pH 3.5 and 9.0, and the absorbed viruses could be eluted with 3% beef extract, pH 9, but not with pH 9.5 to 11.5 glycine-NaOH. Under water quality conditions in which poliovirus recoveries from large volumes of water were less than 5% with conventional negatively charged filters and standard methods, recoveries with Zeta Plus filters averaged 64 and 22.5% for one- and two-stage concentration procedures, respectively. Electropositive filters appear to offer distinct advantages over conventional negatively charged filters for concentrating enteric viruses from water, and their behavior tends to confirm the importance of electrostatic forces in virus recovery from water by microporous filter adsorption-elution methods.     

Concentration of poliovirus from tap water using positively charged microporous filters.

Microporous filters that are more electropositive than the negatively charged filters currently used for virus concentrations from water by filter adsorption-elution methods were evaluated for poliovirus recovery from tap water. Zeta Plus filters composed of diatomaceous earth-cellulose-"charge-modified" resin mixtures and having a net positive charge of up to pH 5 to 6 efficiently adsorbed poliovirus from tap water at ambient pH levels 7.0 to 7.5 without added multivalent cation salts. The adsorbed virus were eluted with glycine-NaOH, pH 9.5 to 11.5. Electropositive asbestos-cellulose filters efficiently adsorbed poliovirus from tap water without added multivalent cation salts between pH 3.5 and 9.0, and the absorbed viruses could be eluted with 3% beef extract, pH 9, but not with pH 9.5 to 11.5 glycine-NaOH. Under water quality conditions in which poliovirus recoveries from large volumes of water were less than 5% with conventional negatively charged filters and standard methods, recoveries with Zeta Plus filters averaged 64 and 22.5% for one- and two-stage concentration procedures, respectively. Electropositive filters appear to offer distinct advantages over conventional negatively charged filters for concentrating enteric viruses from water, and their behavior tends to confirm the importance of electrostatic forces in virus recovery from water by microporous filter adsorption-elution methods.     

Direct force measurements between cellulose surfaces and colloidal silica particles

The interaction of cellulose layers with colloidal silica particles was investigated by direct force measurements with the atomic force microscope (AFM). Upon approach, repulsive forces were found between the negatively charged silica particles and the cellulose surface. The forces were interpreted quantitatively in terms of electrostatic interactions due to overlap of diffuse layers originating from negatively charged carboxylic groups on the cellulose surface. The diffuse layer charge density of cellulose was estimated to be 0.80 mC/m2 at pH 9.5 and 0.21 mC/m2 at pH 4. The forces upon retraction are characterized by molecular adhesion events, whereby individual cellulose chains desorb from the probe surface. The retraction profiles are dominated by well-defined force plateaus, which correspond to single-chain desorption forces of 35-42 pN. We surmise that adsorption of cellulose to probe surfaces is dominated by nonelectrostatic forces, probably originating from hydrogen bonding. Electrostatic contributions to desorption force could be detected only at high pH, where the silica surface is highly charged.     

Some properties of mitochondria, mitoplasts and submitochondrial particles of different polarities from plant tissues

Irrespective of the starting material, i.e. washed mitochondria, purified mitochondria or mitoplast from Solanum tuberosum L., submitochondrial particles of well-defined polarities can be generated by French press treatment in low-salt medium or by sonication in high-salt medium. The first treatment will result in submitochondrial particles which are more than 80% right-side-out (right-side-out particles), the second in submitochondrial particles that are more than 80% inside-out (inside-out particles). The isoelectric point (pI = 4.0) of the inside-out particles measured by cross-partition is distinctly different from the isoelectric points of the other mitochondrial membranes which exhibit pI values between 4.5 and 4.7. The surface charge density measured by 9-aminoacridine fluorescence varies in the same order from −27 mC · m⁻² for Percoll-purified mitochondria to −51 mC · m⁻² for both right-side-out and inside-out particles. Even though the charge densities for the two surfaces of the inner membrane are similar, inside-out particles are much more negatively charged at pH 7.0, since they are 6-times larger. These results clearly demonstrate that it is possible to obtain submitochondrial particles of various polarities and sizes which in turn constitute valuable tools for the study of lateral and transverse asymmetry of the inner mitochondrial membrane.