Aqueous Two-phase Separation Research Articles

Purification of recombinant and human apolipoprotein A-1 using surfactant micelles in aqueous two-phase systems: recycling of thermoseparating polymer and surfactant with temperature-induced phase separation.

An effective system has been developed for purification of apolipoprotein A-1 from Escherichia coli fermentation solution and human plasma using aqueous two-phase extraction and thermal-phase separation. The system included non-ionic surfactants (Triton or Tween) and as top phase-forming polymer a random copolymer of ethylene oxide (50%) and propylene oxide (50%), Breox PAG 50A 1000, was used. The bottom phase-forming polymer was either hydroxypropyl starch, Reppal PES 100 and PES 200, or hydroxyethyl starch, Solfarex A 85. The top-phase-forming polymer and the surfactants are thermoseparating in water solution, i.e., when heated a water phase and a polymer/surfactant phase are formed. Recombinant apolipoprotein A-1, the Milano variant, was extracted from E. coli fermentation solution in a primary Breox-starch phase system followed by thermal separation of the Breox phase where the target protein was recovered in the water phase. Both in the Breox-starch system and in the water-Breox system Triton X-100 was partitioned to the Breox phase. The addition of non-ionic surfactants to the Breox-starch system had strong effect on the purification and yield of the amphiphilic apolipoprotein A-1. In a system containing 17% Breox PAG 50A 1000, 12% Reppal PES 100 and addition of 1% Triton X-100 the purification factor was 7.2, and the yield 85% after thermal separation of the Breox phase. Recycling of copolymer and surfactant was possible after thermal separation of copolymer phase. Approximately 85% of the copolymer and surfactant could be recycled in each extraction cycle. DNA could be strongly partitioned to the starch phase in the primary-phase system. This resulted in a 1000-fold reduction of E. coli DNA in the apolipoprotein A-1 solution obtained after thermoseparation. In extraction from human plasma containing low concentrations of apolipoprotein A-1, it was possible to reach a purification factor of 420 with 98% yield. By reducing the volume ratio to 0.1 Apo A-1 could be concentrated in a small volume of top phase (concentration factor 10) with a yield of 85% and a purification factor of 110.

Aqueous two-phase system of cationic and anionic surfactant mixture and its application to the extraction of porphyrins and metalloporphyrins

Aqueous two-phase system is obtained when a cationic surfactant with bigger alkyl polar head groups such as dodecyltriethylammonium bromide (C 12NE) is mixed with an anionic surfactant like sodium dodecyl sulfate (SDS) at certain surfactant concentrations and molar ratios. When C 12NE is in excess, the top phase is opalescent and is a surfactant-rich micellar solution, while the bottom phase is transparent and is a dilute micellar solution. Such a new aqueous two-phase separation system is employed in extractive separation practice. Several dyes with different chemical structure like anthraquinone, methyl red and methylene blue partitioned in the two-phase system are investigated. Porphyrin compounds and metalloporphyrins, i.e., hematoporphyrin (hemato), uroporphyrin (uro), Cu-TPPS 4 (tetraphenylporphine tetrasulfonic acid), Zn-TPPS 4, Cu-T-4(TAP)P (tetrakis( p-trimethylammoniophenyl) porphine), are also extracted with the present two-phase system. It is found that hydrophobic as well as charge interaction between a substance partitioned in the system and the micelles are predominant factors that govern the extraction.

Tryptophan regulated expression and aqueous two-phase separation of recombinant HIV-fusion peptides

This study investigates the expression and separation of a recombinant HIV-β-gal fusion protein expressed in Escherichia coli under trp promoter control. Product expression and its sensitivity to proteolytic degradation are correlated with inducer strength and protease activity. Two previously unreported proteolytic activities of 42 and 55 kDa molecular weight were revealed which were shown to have activity towards β-gal. Furthermore, by taking advantage of the extreme partitioning behavior of β-galactosidase, the HIV-β-gal fusion was separated and partially purified in a polyethylene glycol-potassium phosphate aqueous two-phase system. Additionally, the 55 kDa protease with β-gal specificity partitioned into the salt-rich bottom phase.

Effects of sugars and polymers on crystallization of poly(ethylene glycol) in frozen solutions: phase separation between incompatible polymers.

This study examined the effect of third components (low-molecular-weight saccharides and polymers) on the crystallization of poly(ethylene) glycol (PEG) in frozen solutions, focusing on the relationship between their crystallization-inhibiting ability and molecular compatibility. Effects of sugars and polymers on the crystallization of PEG 3000 in frozen solution were monitored by differential scanning calorimetry (DSC). Pulsed-NMR was employed to monitor the molecular mobility of water and solutes in the frozen solutions. Miscibility between PEG and third components in aqueous solution was estimated from the lowering of cloud point of PEG 20,000. Thermal analysis of frozen solutions containing some non-crystallizing solutes was used to examine the possibility of phase separation in frozen solutions. Some sugars and polymers inhibited the crystallization of PEG and formed practically stable amorphous phases among ice crystals. The mobility of solute molecules in the amorphous phase increased above the softening temperature of maximally concentrated solutions (Ts), whereas that of water molecules appeared at a lower temperature. Mono- and disaccharides that are relatively less miscible with PEG in solution inhibit PEG crystallization to a lesser degree. Two Ts regions were observed in frozen solutions containing both polyvinylpyrrolidone (PVP) and dextran, at much lower concentrations than those causing aqueous two-phase separation at ambient temperatures. Ice crystallization raises the concentration of solutes in the remaining solution, which can lead to phase separation in the amorphous phase. Molecular compatibility between components is an important factor determining their propensity to phase separate and crystallize.

Preparation of polyacrylamide derivatives showing thermo-reversible coacervate formation and their potential application to two-phase separation processes

A synthetic polymer forming a thermo-reversible coacervate in aqueous milieu was prepared in this study by radical copolymerization of N,N-dimethylacrylamide (DMAA) with N-phenylacrylamide (PA). Increased content of PA in the copolymer (DPA) led to an abrupt decrease in its cloud point (lower critical solution temperature, LCST) owing to an increase in hydrophobicity. The heat of transition ( ΔH) for a 1.0wt% aqueous solution of the copolymer at the LCST was determined to be in the range of 3–4 cal g −1 by differential scanning calorimetry, considerably smaller than that obtained for a neutral amphiphilic polymer, such as poly( N-isopropylacrylamide), undergoing a steep dehydration at the LCST. This small value of ΔH suggests the incomplete dehydration of the polymer chain at the LCST. Indeed, microscopic observation revealed the formation of coacervate droplets in the aqueous solution of the copolymer, which is clear evidence of liquid—liquid phase separation. The concentration of the copolymer in the coacervate phase formed from 1.0wt% aqueous solution of the copolymer at 37°C is approximately 20 wt%. It is of interest that the formation and disappearance of the coacervate are completely thermo-reversible. To estimate the feasibility of applying this thermo-sensitive coacervate system to thermo-modulated aqueous two-phase separation, partitioning of model solutes from aqueous milieu to the coacervate was carried out. By increasing the temperature, preferential partitioning of Trypan Blue (3,3′-[(3,3′-dimethyl[1,1′-biphenyl] 4,4′-diyl)bis(azo)]bis(5-amino-4-hydroxy-2,7-naphthalenedisulfonic acid) tetrasodium salt) from aqueous phase to coacervate phase was observed, whereas no partitioning of vitamin B 12 to coacervate phase took place. Consequently, the separation of Trypan Blue from the mixed solution of vitamin B 12 and Trypan Blue was achieved solely through the change in the environmental temperature. It may be feasible to apply such copolymers showing liquid—liquid phase separation that responds to temperature as the stationary phase in thermo-regulated liquid chromatography for the separation of water-soluble drugs and dyestuffs.

Simultaneous release and purification of gene product in an aqueous two-phase system

For simplification of cell disruption and the subsequent aqueous two-phase separation process, simultaneous release and purification of β-galactosidase from a recombinant Escherichia coli were investigated using a PEG-phosphate aqueous two-phase system containing glycine. The enzyme was released in the two-phase system by the addition of glycine, the percentage of the release increasing with incubation time. The effects of PEG and phosphate concentrations on the release were investigated in a buffer solution. Although the release was enhanced by the addition of PEG, phosphate repressed it. In order to ascertain the optimum operational conditions, the effects of various parameters on the partition coefficient (top/bottom) of the enzyme were investigated. The enzyme was preferentially partitioned to the top phase in a 13% PEG 6000-10% phosphate system, but the partition coefficient was significantly decreased by the addition of NaCl. It was slightly decreased by adding the supernatant of a sonicate of the host cells, but significantly increased with decreasing PEG molecular weight. The addition of glycine to the two-phase system favorably enhanced the partition of the enzyme to the top phase.

Purification and characterization of a strictly specific β- d-fucosidase from Aspergillus phoenicis

Although β- d-fucosidase (β- d-fucohydrolase, EC 3.2.1.38) has been isolated from various sources, all those enzymes were associated with a high activity of β- d-galactosidase and/or β- d-glucosidase. We have purified a specific β- d-fucosidase in electrophoretically homogeneous form from crude extracts of Aspergillus phoenicis by polyethyleneglycol 8000-phosphate buffer aqueous two-phase separation, and successive chromatography on DEAE-Sephadex A-50, hydroxyapatite, and Sephadex G-100 columns. The molecular weight of the enzyme was estimated to be 57,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 50,000 to 60,000 by gel filtration on Sephadex G-100. The enzyme showed optimum activity at pH 6.0 and 40 °C; it was stable in the pH range 5.5–6.5 and below 35 °C. The K m and the V max values for pNP-β - d-fucoside were 2.4 m m, and 12.8 μmol · min −1 · mg −1, respectively. The enzyme was strongly inhibited by sulfhydryl group reagents, p-chloromercuribenzoate, n-ethylmaleimide, and iodoacetate. It was also inhibited by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, diethyl pyrocarbonate, and N-bromosuccinimide. Thus, -SH and -COOH groups and histidyl and tryptophyl residues were essential for enzyme activity. The purified β- d-fucosidase showed high specificity toward p-nitrophenyl-β- d-fucoside. The enzyme was inhibited by d-fucose and d-fucono-γ-lactone, but not by d-galactose, d-galactono-γ-lactone, d-glucose, or d-glucono-γ-lactone; the latter compounds are specific inhibitors of β- d-galactosidase and β- d-glucosidase, respectively. Thus, this enzyme is the most strictly specific β- d-fucosidase when compared with those previously reported.

Magnetic aqueous two-phase separation in preparative applications

Magnetic aqueous two-phase separation is a new technique to speed up the separation of aqueous two-phase systems (Anal. Biochem. 1987, 167, 331–339). It is based on the addition of magnetically susceptible material (e.g. 1-μ iron oxide particles) which induces rapid phase separation when a mixed system is placed in a magnetic field. The technique has been applied to a number of two-phase systems. The time for phase separation was decreased by a factor of 5–240,000, with the largest improvement for systems containing high concentrations of protein and for systems with viscous or nearly isopycnic phases. An apparatus for preparative multistage extraction with magnetic separation was constructed and tested on glycolytic enzymes present in a yeast extract using a dextran/Cibacron blue-polyethylene glycol system. The presence of iron oxide particles did not adversely affect the extracted enzymes. An electromagnet-based apparatus for continuous phase separation on a larger scale was also designed. A phase system containing crude dextran and unpurified cell homogenate was effectively processed. The apparatus also allowed effective separation when the phase containing iron oxide particles was only a small fraction (4%) of the total phase system.

Magnetic aqueous two-phase separation: A new technique to increase rate of phase-separation, using dextran-ferrofluid or larger iron oxide particles

A new technique to speed up the phase separation of aqueous two-phase systems is described. The technique is based on the addition of magnetically susceptible additives (ferrofluids or iron oxide particles). In a magnetic field such additives will induce a faster phase separation. In one approach, dextran-stabilized ferrofluid was added to an aqueous two-phase system containing polyethylene glycol and dextran. The ferrofluid was totally partitioned to the dextran phase. After mixing of the two-phase system, it was possible to reduce the separation time by a factor of 35 by applying a magnetic field to the system. Another approach involved the use of 1-μm iron oxide particles instead of ferrofluid. In this case also, the phase-separation time was reduced, by a factor of about 70, when the system was placed in a magnetic field. The addition of ferrofluid and/or iron oxide particles was shown to have no influence on enzyme partitioning or on enzyme activity. The partitioning of chloroplasts, on the other hand, was influenced unless the ferrofluid used had been treated with epoxysilane. A column system comprising 15 magnetic separation stages was constructed and was used for semicontinuous separation of enzyme mixtures.

Specific binding of 17β-estradiol in the human thymus

Many observations suggest that estrogenic preparations can depress cell-mediated immune reactions. Since cell-mediated immunity is thymus-dependent, the estrogen-binding properties of human and mouse thymus tissue were studied with the use of an aqueous two-phase separation system. A high-affinity and low-capactiy estrogen binding was found in human thymus tissue from six prepubertal children of both sexes and two grown women. A similar specific binding was found in the mouse thymus and in the human, uterus. The association constant (Ka) for the binding in the human thymus was 16.01 ± 8.98 × 109M−1. The human uterus, which is known to possess estrogen receptors, had Ka values in the same range. The binding seems to be located in the reticuloepithelial cells of the thymus. Data suggest that the human thymus is a target organ for estrogens, which may influence lymphocyte function during therapy.

Removal of polyethylene glycol from proteins by salt-induced phase separation.

The use of poly(ethylene glycol) in the purification of plasma and cellular protein is somewhat complicated by the difficulty of removing it from the protein product. The method presented here, quickly and efficiently removes over 95% of the PEG by the simple addition of salts to induce an aqueous two-phase separation with the PEG in the upper phase and greater than 90% of the protein in the lower phase.

Studies on the High Molecular Weight Form of Polypeptide Chain Elongation Factor-1 from Pig Liver

The high molecular weight form of the polypeptide chain elongation factor-1 (EF-1H) from pig liver was purified to an apparently homogeneous state. The procedure included aqueous two-phase separation, ammonium sulfate fractionation, four successive column chromatographies on Sephadex G-200, CM-Sephadex C-50, hydroxylapatite and octyl-Sepharose CL-4B, and glycerol density gradient centrifugation. By this procedure, the specific activity of EF-1H increased about 60-fold with a recovery of the total activity of 6%, as compared to the material obtained by aqueous two-phase separation followed by ammonium sulfate fractionation. Purified EF-1H was analyzed by polyacrylamide gel electrophoresis under non-denaturing conditions and glycerol gradient centrifugation, revealing that it is apparently homogeneous and has both EF-1 alpha and EF-1 beta gamma activities. The subunit structure of EF-1H was investigated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate as well as two-dimensional gel electrophoresis, which indicated that EF-1H consists of three different subunits, i.e., EF-1 alpha, EF-1 beta, and EF-1 gamma, in an equimolar ratio. The Stokes radius and the sedimentation coefficient of EF-1H were estimated to be 5.9 nm and 6.2S, respectively, from which the molecular weight of EF-1H was calculated to be about 157,000. This value is close to the sum of the molecular weights of EF-1 alpha (53,000), EF-1 beta (30,000), and EF-1 gamma (53,000). Thus, we concluded that EF-1H is a monomer of a 1:1:1 complex of EF-1 alpha, EF-1 beta, and EF-1 gamma, or EF-1 alpha beta gamma and its molecular weight is 136,000. This conclusion was further confirmed by measuring the amounts of EF-1 alpha and EF-1 beta gamma in EF-1H by enzymatic assays. The amino acid composition of EF-1H was determined, and the results revealed that there was a close resemblance between the values of EF-1H and those of EF-1 alpha beta gamma, which were calculated from the reported values for EF-1 alpha, EF-1 beta, and EF-1 gamma.

A Simple Method Involving Aqueous Two-Phase Separation-Systems for Activity Determination of Enzymes Hydrolyzing Macromolecular Substrates

Abstract A new general procedure for quantitation of enzymes degrading macromolecular substrates is described. With the use of chromogenic substrates combined with separation of substrate and product by aqueous two-phase systems, rapid separation and accurate measurements can be achieved. Furthermore, in competitive substrate assays using constant amounts of enzyme and chromogenic substrate in combination with a varying amount of native substrate, unknown concentrations of native substrate can easily be determined. The principle is illustrated here by a system consisting of the enzyme, dextranase; the chromogenic substrate, blue dextran; and the native substrate, dextran.

Removal of Polyethylene Glycol from Proteins by Salt-Induced Phase Separation

The use of poly(ethylene glycol) in the purification of plasma and cellular protein is somewhat complicated by the difficulty of removing it from the protein product. The method presented here, quickly and efficiently removes over 95% of the PEG by the simple addition of salts to induce an aqueous two-phase separation with the PEG in the upper phase and greater than 90% of the protein in the lower phase.

Purification and properties of polypeptide chain elongation factor-1alpha from pig liver.

Eukaryotic polypeptide chain elongation factor-1α (EF-1α) has been purified from pig liver by steps including aqueous two-phase separation, ammonium sulfate fractionation, and three successive column chromatographies on CM-Sephadex, DEAE-Sephadex, and CM-Sephadex. On the last column chromatography on CM-Sephadex, EF-1α activity was eluted as four peaks. These peaks except the first one appear to be homogeneous as judged by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and isoelectric focusing in polyacrylamide gel. There was no difference between these four peaks in the activity to promote the binding of Phe-tRNA to ribosomes in the presence of guanyl-5′-yl methylenediphosphonate. They had similar molecular weights and similar amino acid compositions but different isoelectric points which varied from 9.3 to 9.9. The molecular weight of EF-1α was estimated to be 53,000 and 61,000 by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and by gel filtration on Sephadex G-150, respectively, indicating that EF-1α is composed of a single polypeptide. EF-1α appears to contain 6 half-cystine residues per molecule, at least one of which is essential for its activity. The effect of the concentration of NH4 on the dissociation constant of the binary complex containing EF-lα and guanine nucleotides (EF-1α.GDP or EF-1α.GTP) was extensively investigated, and it was found that the logarithm of the dissociation constant of EF-1α.GDP increased proportionally to the square root of the concentration of NH4Cl, while that of EF-1α.GTP decreased. Similar relationships were observed between the rate constant of dissociation of EF-1α.GDP or EF-1α.GTP and the concentration of NH4 When the rate constant of association was calculated from these values, the logarithm of the constant of EF-1α.GDP and that of EF-1α.GTP found to decrease proportionally to the square root of the NH4Cl concentration.

Purification of polyhedra of a cytoplasmic polyhedrosis virus from soil using metrizamide

T. Hukuhara (J. Invertebr. Pathol., 25, 337-342, 1975) developed a method of purifying virus polyhedra from the soil based on the following three steps: (1) desorption of polyhedra with sodium pyrophosphate; (2) aqueous two-phase separation; and (3) CsCl density-gra~ent cen~ifugation. The recovery of polyhedra by this method was low because of incomplete desorption in step 1 and soil aggregation caused by the high ionicity in step 3. If the method is used for field application, large amounts of soil will be needed because the soil is likely to contain only a relatively small amount of virus, and the sample must be concentrated prior to step 3 to reduce the sample volume to accommodate for high-speed centrifugation. We have investigated the effect of ultrasonication in step 1, the procedures for concentrating the sample obtained in step 2, and the use of a nonionic gradient material in step 3. Polyhedra of a cytoplasmic polyhedrosis virus of Bombyx mori were thoroughly mixed with brown forest soil (log polyhedra/g of soil). The preparation of the mixture, the desorption of polyhedra, and the separation by a biphasic system were performed as in the original method, except that some samples were ultrasonicated for 1 hr at 30 kc (700 W) in an ultrasonic oscillator (Toyo Riko Seisakusho, Tokyo, Type UD-N-50-6) before being incorporated into the biphasic system (Table 1). Contrary to expectation, both the recovery rate of polyhedra and the purification factor were lower when the samples were ultrasonicated (Table 2). The ultrasonicated samples contained a higher proportion of clay particles because of the decomposition of the larger soil particles. This resulted in a ninefold increase in the amount of soil entering the upper phase of the biphasic system. The reduced recovery rate of polyhedra was presumably due to the increased soil surface which adsorbed the polyhedra. After biphasic separation, the upper phase was diluted with an equal volume of distilled water and was centrifuged for 1 hr at 1800 rpm (65Og) to sediment the soil and polyhedra. The pellet was resuspended in a small volume of distilled water, about one-eighteenth of the original volume of the upper phase and was thoroughly mixed with an equal volume of an 80% (w/v) solution of metrizamide [2-(3-acetamido-5-N-methylacetamido 2,4,6 triiodobenzamido) 2 deoxy-D-glucose] (Nyegaard & Co. A/S, Oslo, Norway) in 0.2 M Tris buffer,pH 7.0. Some samples were combined with a CsCl solution as in the original method and served as controls. After centrifugation of the nonultrasonicated samples for 20 hr at 60,000 rpm (314,000g at R,,,) in a Beckman fixedangle rotor (Type 65Y), a sharp white band of polyhedra appeared in the middle of the metrizamide density-gradient column and at the top of the CsCl column. When ultrasonicated samples were used, the bands were more diffuse, and the number of polyhedra recovered was less in the metrizamide column but nearly the same in the CsCl column. The best recovery of purified polyhedra was obtained when the nonultrasonicated samples were centrifuged in the metrizamide column (Table 2). We tested two other biphasic systems for their efficiencies in concentrating the sample prior to step 3, as compared to the simple centrifugation described above. When the upper phase obtained in step 2 was mixed with l/100 vol of 20% dextran sulfate 500 (Pharmacia, Uppsala, Sweden) and l/l0 vol of 3 M NaCl and was placed overnight in the cold, the mixture separated into two phases, an upper polyethylene

Purification and properties of a new polypeptide chain elongation factor, EF-1beta, from pig liver.

Eukaryotic polypeptide elongation factor 1 (EF-1) from pig liver has been resolved into two complementary factors, EF-1alpha and EF-1beta (Iwasaki, K., Mizumoto, K., Tanka, M., and Kaziro, Y. (1973) J. Biochem. (Tokyo) 74, 849). This paper describes the procedures for purification of EF-1beta and some properties of the purified factor. The purification method includes an aqueous two-phase separation technique, a treatment of the crude factor with sodium cholate and two successive column chromatographies on diethyl-aminoethyl-Sephadex A-50. By this method, EF-1beta was purified about 50-fold starting from the material obtained after two-phase separation followed by ammonium sulfate fractionation with a recovery of 20%. The purified EF-1beta appeared homogeneous, having a molecular weight of about 90,000. It consisted of two unequal subunits of the molecular weights of 55,000 and 30,000. It stimulates polymerization of phenylalanine dependent on poly(U) in the presence of both EF-1alpha and EF-2, as well as the EF-1alpha-dependent binding of phenylalanyl-tRNA to ribosomes in the presence of GTP. However, it had no effect on the stoichiometric binding of phenylalanyl-tRNA to ribosomes dependent on EF-1alpha in the presence of guanyl-5'-yl methylenediphosphonate. These results indicate that the function of EF-1beta is to stimulate the recycling of EF-1alpha.

Purification of polyhedra of a cytoplasmic-polyhedrosis virus from soil

Polydedra of a cytoplasmic-polyhedrosis virus of the silkworm, Bombyx mori, were purified from soil by multistep procedures consisting of desorption of the polyhedra with sodium pyrophosphate, aqueous two-phase separation with the dextran-polyethylene glycol system, and CsCl density-gradient centrifugation. The overall recovery of polyhedra was 7% and there was a 170-fold increase in the number of polyhedra per gram minerals.

The Purification of Low Molecular Weight Form of Polypeptide Elongation Factor 1 from Pig Liver

Abstract Eukaryotic polypeptide elongation factor 1 (EF-1) from pig liver has recently been resolved into two complementary factors, EF-1α and EF-1β (Iwasaki, K., Mizumoto, K., Tanaka, M., and Kaziro, Y. (1973) J. Biochem. (Tokyo) 74, 849). This paper describes a new procedure for the purification of low molecular weight form of EF-1α from pig liver. The new purification method includes an aqueous two-phase separation technique and two successive column chromatographic steps on carboxymethyl-Sephadex C-50. By this method, EF-1α was purified about 450-fold starting from the postmitochondrial fraction of pig liver homogenates with a recovery of 26%. The purified EF-1α appeared to be nearly homogeneous and was shown to consist of a single polypeptide with a molecular weight of 53,000. It promotes the synthesis of polyphenylalanine in the presence of added polypeptide elongation factor 2 (EF-2) and EF-1β, as well as the poly(U)-dependent binding of phenylalanyl-tRNA to ribosomes with either GTP or 5'-guanylyl methylenediphosphonate in the absence of both EF-1β and EF-2. Its specific activity measured by poly(U)-dependent polyphenylalanine synthesis from phenylalanyl-tRNA has been estimated to be 423 nmoles of phenylalanine polymerized per mg of protein during a 30-min incubation at 37°.

Virus in water. II. Evaluation of membrane cartridge filters for recovering low multiplicities of poliovirus from water.

The efficiency of a Millitube MF cartridge filter, a membrane filter, for recovery of poliovirus from 100-gal volumes of both fresh (tap) and estuarine water was determined. In the high multiplicity of virus input-output experiments, recovery of 97% or greater of input virus was achieved in both types of water when the final concentration of divalent cation as Mg(2+) was 1,200 mug/ml and the pH was 4.5. Virus was effectively eluted from the membrane cartridge with 5x nutrient broth in 0.05 M carbonate-bicarbonate buffer at pH 9.0. Four elutions of 250 ml each were used. In the low multiplicity of virus input-output experiments under the same cationic and pH conditions, up to 67% of the input virus was recovered when the virus was further concentrated from the eluates by the aqueous polymer two-phase separation technique. The volume reduction was 126,000-190,000 to 1. The use of the combined techniques, i.e., membrane adsorption followed by aqueous polymer two-phase separation, provided a highly sensitive, simple, and remarkably reliable sequential methodology for the quantitative recovery of poliovirus occurring at multiplicities as low as 1 to 2 plaque-forming units per 5 gal of water.