Concentration and purification of rubella virus hemagglutinin by hollow fiber ultrafiltration and sucrose density centrifugation

1980 ◽  
Vol 26 (11) ◽  
pp. 1334-1339 ◽  
Author(s):  
M. Trudel ◽  
P. Payment
Keyword(s):  
Molecular Weight ◽  
Tissue Culture ◽  
Vero Cell ◽  
Hollow Fiber ◽  
Rubella Virus ◽  
Specific Activity ◽  
Sucrose Density Gradient ◽  
Hollow Fibers ◽  
Cell Monolayers ◽  
Molecular Weight Cutoff

Large volumes of rubella virus were produced in Vero cell monolayers which were grown in the Corbeil-Bellco TM system. Infectious tissue culture fluids were concentrated at least 600 times in less than 4 h by ultrafiltration on hollow fibers with a molecular weight cutoff of 100 000. Recovery of the hemagglutinating activity was 75%. Rubella virus was purified by three successive sucrose density gradient centrifugations using a combination of discontinuous and linear gradients. Specific activity was increased 1000-fold.

Advances in the use of low-pressure, hollow fiber membranes for the disinfection of water

2005 ◽  
Vol 5 (5) ◽  
pp. 109-115 ◽  
Author(s):  
J.G. Jacangelo ◽  
A. Madec ◽  
K.J. Schwab ◽  
D.E. Huffman ◽  
C.S. Mysore
Keyword(s):  
Molecular Weight ◽  
Pore Size ◽  
Hollow Fiber ◽  
Good Predictor ◽  
Low Pressure ◽  
Hollow Fiber Membranes ◽  
Virus Removal ◽  
Testing Unit ◽  
Bench Scale ◽  
Molecular Weight Cutoff

Low-pressure membranes are often used for disinfection purposes. Using a simple bench-scale testing unit, several membranes were evaluated for their capability to remove microorganisms under specific conditions. Five hollow fiber modified polysulfone membranes ranging in molecular weight cutoff (MWCO) from 10,000 to 300,000 daltons and a 0.2 μm nominal pore size membrane were also evaluated. The 10,000 MWCO membrane removed less virus than the two 100,000 MWCO or the 300,000 MWCO membranes. The data from this study suggests that MWCO and specific flux may not, in all cases, be a good predictor of virus removal.

Aggregation States of a Regulatory Enzyme Complex Catalyzing the Early Steps of Pyrimidine Biosynthesis in Bakers' Yeast

1971 ◽  
Vol 49 (4) ◽  
pp. 403-411 ◽  
Author(s):  
P. F. Lue ◽  
J. G. Kaplan
Keyword(s):  
Molecular Weight ◽  
Density Gradient ◽  
Crude Extract ◽  
Analytical Ultracentrifugation ◽  
Specific Activity ◽  
Feedback Inhibition ◽  
Gradient Centrifugation ◽  
Sucrose Density Gradient ◽  
Regulatory Site ◽  
Aspartate Transcarbamylase

Aspartate transcarbamylase (ATCase) of bakers' yeast has been purified 78-fold from a crude extract of a derepressed diploid strain; its specific activity was more than 300-fold that of a wild-type crude extract. During the last steps of the purification there was a parallel co-purification of carbamylphosphate synthetase (CPSase), and both activities retained full sensitivity to feedback inhibition by UTP; indeed the sensitivity of the ATCase to UTP increased during the purification doubtless due to discard of a feedback-insensitive ATCase subunit. The two enzyme activities co-eluted from gel filtration on Sepharose 6B together with the feedback site. Analytical ultracentrifugation revealed that the material was not homogeneous, showing two major peaks. Sucrose density gradient centrifugation in the presence of UTP, glutamine, and Mg2+ resulted in co-sedimentation of the two activities and the regulatory site, corresponding to a molecular weight of approximately 800 000 daltons. Omission of UTP from the gradient resulted in disappearance of the heavy peak and appearance of a new one, corresponding to a molecular weight of 380 000 and possessing both activities; the CPSase was still highly sensitive to UTP unlike the ATCase which was only slightly sensitive to retroinhibition. Omission of glutamine and Mg2+ from the sucrose density gradient caused a distinct CPSase peak to trail behind the ATCase; again, the CPSase (molecular weight 250 000) retained full sensitivity to feedback inhibition. This, together with genetic data, supports the view that the ura-2 gene which controls ATCase, CPSase, and the regulatory site is a polycistronic operon, coding for the production of two or three polypeptide chains; the CPSase subunit is inactive unless a regulatory site is present, whereas the ATCase subunit (molecular weight 140 000) is highly active but completely insensitive to feedback inhibition.

Preparation and Properties of Human Prothrombin Complex

1970 ◽  
Vol 24 (03/04) ◽  
pp. 325-333 ◽  
Author(s):  
G. H Tishkoff ◽  
L. C Williams ◽  
D. M Brown
Keyword(s):  
Molecular Weight ◽  
Proteolytic Enzyme ◽  
Enzyme Inhibitor ◽  
Specific Activity ◽  
Crystalline Form ◽  
Sedimentation Equilibrium ◽  
Crystalline Complex ◽  
Interaction Product ◽  
Proteolytic Enzyme Inhibitor ◽  
Purification Scheme

SummaryAs a corollary to our previous studies with bovine prothrombin, we have initiated a study of human prothrombin complex. This product has been isolated in crystalline form as a barium glycoprotein interaction product. Product yields were reduced compared to bovine product due to the increased solubility of the barium glycoprotein interaction product. On occasion the crystalline complex exhibited good yields. The specific activity of the crystalline complex was 1851 Iowa u/mg. Further purification of human prothrombin complex was made by removal of barium and by chromatography on Sephadex G-100 gels. The final product evidenced multiple procoagulant activities (II, VII, IX and X). The monomeric molecular weight determined by sedimentation equilibrium in a solvent of 6 M guanidine-HCl and 0.5% mercaptoethanol was 70,191 ± 3,057 and was homogeneous with respect to molecular weight. This product was characterized in regard to physical constants and chemical composition. In general, the molecular properties of human prothrombin complex are very similar to the comparable bovine product. In some preparations a reversible proteolytic enzyme inhibitor (p-aminophenylarsonic acid) was employed in the ultrafiltration step of the purification scheme to inhibit protein degradation.

Enterovirus Concentration Using Automated Hollow Fiber Ultrafiltration

1982 ◽  
Vol 14 (4-5) ◽  
pp. 257-272 ◽  
Author(s):  
G Belfort ◽  
A Paluszek ◽  
L S Sturman
Keyword(s):  
Drinking Water ◽  
Hollow Fiber ◽  
Enhanced Recovery ◽  
Tap Water ◽  
Membrane Surface ◽  
Creeping Flow ◽  
Hollow Fibers ◽  
Virus Concentration ◽  
Concentration Technique ◽  
Solution Interface

The Automated Hollow Fiber Ultrafiltration (AHFU) method is proposed here as a simple, efficient and rapid virus concentration technique from tap and drinking water sources. The results reported here extend the testing of the AHFU method to include two Picornaviruses [Poliovirus 2 (vaccine) and Echovirus 1] and Reovirus 3. Their respective mean virus recoveries from between 3 and 100 l of tap water is 88 ± 26, 79 ± 60, and 104 ± 48%. Various approaches including membrane surface modification, changes in backwash hydrodynamics, modification of the feed and backwash composition, and the use of S35-methionine labelled Poliovirus 2, are used to study the recovery of sorbed Poliovirus 2 from the hollow fiber/solution interface. An increase in the backwash pH to between 9.5 and 10.5 significantly improved Poliovirus 2 recovery. This, together with the labelled experiments, indicates that the virus-membrane interactions are probably electrostatic in nature. Convective polarization during filtration probably brings the virus close enough to the surface for these interactions to occur since virus losses were not detected for a non-permeation recycle experiment. Because very low Reynold's numbers are used, the flow is in the creeping-flow-regime for both filtration and backwashing (axial and radial). Unless significantly higher Reynolds could be used, enhanced recovery due to purely hydrodynamic forces is unlikely. High Reynold's numbers, of course, are limited by the pressure constraints of the hollow fibers.

Existence of lipid vesicles containing platelet-activating factor in endothelial cell lysate

1992 ◽  
Vol 12 (1) ◽  
pp. 15-21
Author(s):  
S. Kojima ◽  
K. Nara ◽  
Y. Inada ◽  
S. Hirose ◽  
Y. Saito
Keyword(s):  
Molecular Weight ◽  
Endothelial Cell ◽  
Specific Activity ◽  
Gel Filtration ◽  
Platelet Activating Factor ◽  
Lipid Vesicles ◽  
Specific Factor ◽  
Cell Lysate ◽  
Aggregation Activity ◽  
Molecular Weight Fractions

Platelet aggregation activity due to platelet-activating factor (PAF) was detected at high molecular weight (HMW) and low molecular weight fractions after gel-filtration chromatography of cell lysate of endothelial cells. [3H]PAF added to the cell lysate was similarly distributed after chromatography. The radioactivity associated with HMW fraction was not reduced by digesting the lysate with trypsin, suggesting that PAF was not making complexes with proteins but was included in lipid vesicles in cell lysate. Further evidence showed that an unknown specific factor(s) was needed to form these PAF-containing lipid vesicles. Radioactivity was not found in HMW fraction when [3H]PAF was mixed with cell lysate of vascular smooth muscle cells. When monomeric PAF was added to endothelial cell lysate, the specific activity of aggregation decreased to the level exerted by endogenous PAF-containing lipid vesicles due to incorporation into lipid vesicles. PAF in the form of lipid vesicles was more stable in plasma than monomeric form.

scholarly journals Hydrophilic Dual Layer Hollow Fiber Membranes for Ultrafiltration

Membranes ◽  
2020 ◽  
Vol 10 (7) ◽  
pp. 143
Author(s):  
Lara Grünig ◽  
Ulrich A. Handge ◽  
Joachim Koll ◽  
Oliver Gronwald ◽  
Martin Weber ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Hollow Fiber ◽  
Triblock Copolymer ◽  
Pure Water ◽  
Vinyl Pyrrolidone ◽  
Scattering Data ◽  
Coupling Mechanism ◽  
Additive Concentration ◽  
Hollow Fiber Membranes ◽  
Fiber Membranes

In this study, a triblock copolymer was used as additive to fabricate new dual layer hollow fiber membranes with a hydrophilic active inner surface in order to improve their fouling resistance. The polymeric components of the solutions for membrane fabrication were poly(ether sulfone), poly(N-vinyl pyrrolidone), and the triblock copolymer. The additive consists of three blocks: a middle hydrophobic poly(ether sulfone) block and two outer hydrophilic alkyl poly(ethylene glycol) blocks. By varying the additive concentration in the solutions, it was possible to fabricate dual layer hollow fiber membranes that are characterized by a hydrophilic inner layer, a pure water permeance of over 1800 L/(m2 bar h) and a molecular weight cut-off of 100 kDa similar to commercial membranes. Contact angle and composition determination by XPS measurements revealed the hydrophilic character of the membranes, which improved with increasing additive concentration. Rheological, dynamic light scattering, transmission, and cloud point experiments elucidated the molecular interaction, precipitation, and spinning behavior of the solutions. The low-molecular weight additive reduces the solution viscosity and thus the average relaxation time. On the contrary, slow processes appear with increasing additive concentration in the scattering data. Furthermore, phase separation occurred at a lower non-solvent concentration and the precipitation time increased with increasing additive content. These effects revealed a coupling mechanism of the triblock copolymer with poly(N-vinyl pyrrolidone) in solution. The chosen process parameters as well as the additive solutions provide an easy and inexpensive way to create an antifouling protection layer in situ with established recipes of poly(ether sulfone) hollow fiber membranes. Therefore, the membranes are promising candidates for fast integration in the membrane industry.

scholarly journals CONCENTRATION AND PURIFICATION OF BACTERIOPHAGE

1938 ◽  
Vol 21 (3) ◽  
pp. 335-366 ◽  
Author(s):  
John H. Northrop
Keyword(s):  
Molecular Weight ◽  
Small Molecules ◽  
Specific Activity ◽  
Active Agent ◽  
Pure Substance ◽  
Solubility Curve ◽  
Denatured Protein ◽  
Sedimentation Constant ◽  
Rate Of Sedimentation ◽  
Living Organisms

1. A method for isolating a nucleoprotein from lysed staphylococci culture is described. 2. It is homogeneous in the ultracentrifuge and has a sedimentation constant of 650 x 10–13 cm. dyne–1 sec.–1, corresponding to a molecular weight of about 300,000,000. 3. The diffusion coefficient varies from about 0.001 cm.2/day in solutions containing more than 0.1 mg. protein/ml. to 0.02 in solutions containing less than 0.001 mg. protein/ml. The rate of sedimentation also decreases as the concentration decreases. It is suggested, therefore, that this protein exists in various sized molecules of from 500,000–300,000,000 molecular weight, the proportion of small molecules increasing as the concentration decreases. 4. This protein is very unstable and is denatured by acidity greater than pH 5.0, by temperature over 50°C. for 5 minutes. It is digested by chymo-trypsin but not by trypsin. 5. The loss in activity by heat, acid, and chymo-trypsin digestion is roughly proportional to the amount of denatured protein formed under these conditions. 6. The rate of diffusion of the protein is the same as that of the active agent. 7. The rate of sedimentation of the protein is the same as that of the active agent. 8. The loss in activity when susceptible living or dead bacteria are added to a solution of the protein is proportional to the loss in protein from the solution. Non-susceptible bacteria remove neither protein nor activity. 9. The relative ultraviolet light absorption, as determined directly, agrees with that calculated from Gates' inactivation experiments in the range of 2500–3000 Å. u. but is somewhat greater in the range of 2000–2500 Å. u. 10. Solubility determinations showed that most of the preparations contained at least two proteins, one being probably the denatured form of the other. Two preparations were obtained, however, which had about twice the specific activity of the earlier ones and which gave a solubility curve approximating that of a pure substance. 11. It is suggested that the formation of phage may be more simply explained by analogy with the autocatalytic formation of pepsin and trypsin than by analogy with the far more complicated system of living organisms.

RUBELLA VIRUS IN TISSUE-CULTURE

The Lancet ◽  
1954 ◽  
Vol 264 (6848) ◽  
pp. 1107-1108 ◽  
Author(s):  
S ANDERSON
Keyword(s):  
Tissue Culture ◽  
Rubella Virus
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