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.

FURTHER STUDIES ON THE PURIFICATION OF THROMBIN

1958 ◽  
Vol 36 (1) ◽  
pp. 603-611 ◽  
Author(s):  
Walter H. Seegers ◽  
Walter G. Levine ◽  
Robert S. Shepard
Keyword(s):  
Molecular Weight ◽  
Phosphate Buffer ◽  
Esterase Activity ◽  
Room Temperature ◽  
Specific Activity ◽  
Dry Weight ◽  
The Other ◽  
Resin Column ◽  
Sedimentation Constant ◽  
Single Symmetrical Peak

Purified biothrombin (bovine) was fractionated with the use of amberlite IRC-50 columns to obtain resin thrombin with an activity of 4100 units/mg. dry weight or 45,000 units/mg. tyrosine. As obtained from a resin column in 0.3 M phosphate buffer, pH 8.0, the thrombin is stable for 5 days at room temperature. At 4 °C. about 70% of the activity remains after 20 weeks. The maximum molecular weight is estimated by comparing with the specific activity (2000 units/mg.) and molecular weight (62,700) of purified prothrombin as follows: 2000/4100 × 62,700 or 30,600 as the probable molecular weight. Resin thrombin can lose its fibrinogen-clotting power while esterase activity is retained. On the other hand the esterase activity can be depressed without diminishing the clotting activity. Resin thrombin lyses fibrin. When examined in an ultracentrifuge a single symmetrical peak was found with a sedimentation constant of S = 3.9 (20 °C., 0.1 M KCl, 5.5 mg./ml.) Citrate thrombin was also fractionated with the use of IRC-50 to obtain material with a specific activity of 47,000 units/mg. tyrosine.

FURTHER STUDIES ON THE PURIFICATION OF THROMBIN

1958 ◽  
Vol 36 (6) ◽  
pp. 603-611 ◽  
Author(s):  
Walter H. Seegers ◽  
Walter G. Levine ◽  
Robert S. Shepard
Keyword(s):  
Molecular Weight ◽  
Phosphate Buffer ◽  
Esterase Activity ◽  
Room Temperature ◽  
Specific Activity ◽  
Dry Weight ◽  
The Other ◽  
Resin Column ◽  
Sedimentation Constant ◽  
Single Symmetrical Peak

Purified biothrombin (bovine) was fractionated with the use of amberlite IRC-50 columns to obtain resin thrombin with an activity of 4100 units/mg. dry weight or 45,000 units/mg. tyrosine. As obtained from a resin column in 0.3 M phosphate buffer, pH 8.0, the thrombin is stable for 5 days at room temperature. At 4 °C. about 70% of the activity remains after 20 weeks. The maximum molecular weight is estimated by comparing with the specific activity (2000 units/mg.) and molecular weight (62,700) of purified prothrombin as follows: 2000/4100 × 62,700 or 30,600 as the probable molecular weight. Resin thrombin can lose its fibrinogen-clotting power while esterase activity is retained. On the other hand the esterase activity can be depressed without diminishing the clotting activity. Resin thrombin lyses fibrin. When examined in an ultracentrifuge a single symmetrical peak was found with a sedimentation constant of S = 3.9 (20 °C., 0.1 M KCl, 5.5 mg./ml.) Citrate thrombin was also fractionated with the use of IRC-50 to obtain material with a specific activity of 47,000 units/mg. tyrosine.

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.

Conjugates of Classical DNA/RNA Binder with Nucleobase: Chemical, Biochemical and Biomedical Applications

2019 ◽  
Vol 26 (30) ◽  
pp. 5609-5624
Author(s):  
Dijana Saftić ◽  
Željka Ban ◽  
Josipa Matić ◽  
Lidija-Marija Tumirv ◽  
Ivo Piantanida
Keyword(s):  
Molecular Weight ◽  
Small Molecules ◽  
Biomedical Applications ◽  
Protein Targets ◽  
New Class ◽  
Rna Targets ◽  
Covalently Linked ◽  
Structural Aspects

: Among the most intensively studied classes of small molecules (molecular weight < 650) in biomedical research are small molecules that non-covalently bind to DNA/RNA, and another intensively studied class is nucleobase derivatives. Both classes have been intensively elaborated in many books and reviews. However, conjugates consisting of DNA/RNA binder covalently linked to nucleobase are much less studied and have not been reviewed in the last two decades. Therefore, this review summarized reports on the design of classical DNA/RNA binder – nucleobase conjugates, as well as data about their interactions with various DNA or RNA targets, and even in some cases protein targets are involved. According to these data, the most important structural aspects of selective or even specific recognition between small molecule and target are proposed, and where possible related biochemical and biomedical aspects were discussed. The general conclusion is that this, rather new class of molecules showed an amazing set of recognition tools for numerous DNA or RNA targets in the last two decades, as well as few intriguing in vitro and in vivo selectivities. Several lead research lines show promising advancements toward either novel, highly selective markers or bioactive, potentially druggable molecules.

scholarly journals Functionalization Strategies of PEDOT and PEDOT:PSS Films for Organic Bioelectronics Applications

Chemosensors ◽  
2021 ◽  
Vol 9 (8) ◽  
pp. 212
Author(s):  
Gonzalo E. Fenoy ◽  
Omar Azzaroni ◽  
Wolfgang Knoll ◽  
Waldemar A. Marmisollé
Keyword(s):  
Small Molecules ◽  
Conducting Polymer ◽  
Organic Semiconductors ◽  
Synthesis Method ◽  
Polymer Synthesis ◽  
Preparation Methods ◽  
Electrophysiological Signals ◽  
Organic Bioelectronics ◽  
Biological Interfaces ◽  
Living Organisms

Organic bioelectronics involves the connection of organic semiconductors with living organisms, organs, tissues, cells, membranes, proteins, and even small molecules. In recent years, this field has received great interest due to the development of all kinds of devices architectures, enabling the detection of several relevant biomarkers, the stimulation and sensing of cells and tissues, and the recording of electrophysiological signals, among others. In this review, we discuss recent functionalization approaches for PEDOT and PEDOT:PSS films with the aim of integrating biomolecules for the fabrication of bioelectronics platforms. As the choice of the strategy is determined by the conducting polymer synthesis method, initially PEDOT and PEDOT:PSS films preparation methods are presented. Later, a wide variety of PEDOT functionalization approaches are discussed, together with bioconjugation techniques to develop efficient organic-biological interfaces. Finally, and by making use of these approaches, the fabrication of different platforms towards organic bioelectronics devices is reviewed.

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 Correlations between retention volume and molecular size parameters in gel permeation chromatography of small molecules

1975 ◽  
Vol 28 (1) ◽  
pp. 189 ◽  
Author(s):  
RA Shanks
Keyword(s):  
Molecular Weight ◽  
Small Molecules ◽  
Organic Molecules ◽  
Retention Volume ◽  
Molecular Size ◽  
Gel Permeation Chromatography ◽  
Molar Volumes ◽  
Small Organic Molecules ◽  
Molecular Dimension ◽  
Gel Permeation

Gel permeation columns of Bio Beads S-X8 have been used to provide separation of oligomers and other small organic molecules. Results show successful separations up to molecular weight c. 600. The retention times of compounds have been correlated with the largest molecular dimension of the molecules and also with molar volumes.

scholarly journals The Nature of the Muscle-Relaxing Factor

1963 ◽  
Vol 46 (5) ◽  
pp. 893-904 ◽  
Author(s):  
Franklin Fuchs ◽  
F. Norman Briggs
Keyword(s):  
Molecular Weight ◽  
High Speed ◽  
Gel Filtration ◽  
Active Agent ◽  
Calcium Deficiency ◽  
Chelating Resin ◽  
Prolonged Heating ◽  
Relaxing Factor ◽  
Chelex 100 ◽  
High Molecular Weight Material

High speed centrifugal fractionation of homogenates of rabbit skeletal muscle has led to the discovery of a soluble muscle-relaxing factor in the homogenate. Assay of the relaxing activity with deoxycholate-treated myofibrils and reconstituted actomyosin systems has established that the activity is not produced by the presence of contaminants. Relaxing activity could be removed or destroyed by charcoal, dialysis, prolonged heating, and treatment with the chelating resin, chelex-100, making it improbable that the effect is due simply to calcium deficiency. Many of the characteristics of this muscle-relaxing factor suggest that it is very similar to or the same as the factor formed by the incubation of muscle granule fractions and ATP. Evidence is presented that some soluble protein component is involved in the stabilization of the factor. The relaxing activity could be separated from the high molecular weight material in the supernatant by the technique of gel filtration. On the basis of the gel used, the molecular weight of the active agent should be less than 4000.

scholarly journals MOLECULAR WEIGHT AND ELECTROPHORESIS OF CRYSTALLINE RIBONUCLEASE

1940 ◽  
Vol 24 (2) ◽  
pp. 203-211 ◽  
Author(s):  
Alexandre Rothen
Keyword(s):  
Molecular Weight ◽  
Isoelectric Point ◽  
Unit Weight ◽  
Diffusion Measurement ◽  
Average Value ◽  
Rate Of Sedimentation ◽  
And Diffusion ◽  
Good Agreement

Electrophoretic studies on purified crystalline ribonuclease showed the absence of any impurities differing in mobility from the bulk of material. The isoelectric point of ribonuclease was found by electrophoresis to be at about pH 7.8. Ultracentrifuge studies indicated fair homogeneity of ribonuclease in solution. Only one moving component has been observed. The molecular weight of ribonuclease was found to be 12,700 from rate of sedimentation (S25 = 1.85 x 10–13 in 0.5 M (NH4)2SO4) and diffusion measurement (D = 1.36 x 10–6 in 0.5 M (NH4)2SO4), in good agreement with the average value of 13,000 found from equilibrium measurements. This low value for the molecular weight of a protein would seem to discredit the value 17,600 as representing a universal unit weight for proteins in general.

Novel Catalatic Proteins of Bakers' Yeast. I. An Atypical Catalase

1973 ◽  
Vol 51 (11) ◽  
pp. 1551-1555 ◽  
Author(s):  
Tony C. M. Seah ◽  
A. R. Bhatti ◽  
J. G. Kaplan
Keyword(s):  
Molecular Weight ◽  
Gel Electrophoresis ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Native Protein ◽  
Wild Type ◽  
Major Band ◽  
Subunit Molecular Weight ◽  
Purification And Properties

At any stage of growth of a wild-type bakers' yeast, some 20% of the catalatic activity of crude extracts is not precipitable by means of antibody prepared against the typical catalase (catalase T), whose purification and properties have been previously described. Some of this catalatic activity is due to the presence of an atypical catalase (catalase A), a heme protein, with a molecular weight estimated as 170 000 – 190 000, considerably lower than that of the usual catalases (225 000 – 250 000). Preparations of catalase A were found to be homogeneous in the analytical ultracentrifuge and in polyacrylamide gel electrophoresis. Its subunit molecular weight, determined from its iron content, was 46 500, virtually the same as that of the major band obtained in gel electrophoresis in the presence of sodium dodecyl sulfate, suggesting that the native protein is tetrameric. Its specific activity is in the range of those reported for other typical catalases.

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