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.

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.

PROPERTIES OF BOVINE Ac-GLOBULIN CONCENTRATES AND METHODS OF PREPARATION

1963 ◽  
Vol 41 (1) ◽  
pp. 2409-2421 ◽  
Author(s):  
Nobuo Aoki ◽  
Charles R. Harmison ◽  
Walter H. Seegers
Keyword(s):  
Human Plasma ◽  
Ammonium Sulphate ◽  
Protein Concentration ◽  
Room Temperature ◽  
Specific Activity ◽  
Barium Carbonate ◽  
Dry Weight ◽  
Physical Chemical ◽  
Bovine Plasma ◽  
Refrigerator Temperature

A procedure is described for retaining bovine plasma Ac-globulin activity as one part of the protein from plasma for every 1000 parts removed. The yields averaged 15%. The procedure involves removal of prothrombin with barium carbonate, isoelectric fractionation, fractionation with ammonium sulphate, chromatography on Amberlite IRC-50, and a second fractionation with ammonium sulphate. The procedure requires 2 days; however, the first day completes up to chromatography and the concentrate at that time is quite useful for many purposes. It is more stable than the product obtained after chromatography and the yields are higher. In absence of salts Ac-globulin is quite insoluble at pH 5.0. The final product usually contained some impurity. With the analytical ultra-centrifuge the S20in 0.1 M potassium chloride solution was found to be 4.2 at a protein concentration of 12.4 mg/ml. The specific activity was 1500 U./mg dry weight. Bovine plasma contains 120 U./ml or about 9 mg/100 ml. Assuming the same specific activity for human plasma the concentration is most likely near 1 mg/100 ml. The best stability conditions found were: 50% glycerol, pH 7.0, and 0.1 M calcium chloride. Under those conditions at room temperature all activity was retained 6 to 7 hours, at refrigerator temperature 24 hours, and at −60 °C for 1 month. In rabbits, antibodies were readily produced. Oxidizing agents destroyed the activity, while reducing agents did not, nor did they tend to stabilize. SH blocking agents destroyed the activity. The loss of activity in the presence of 0.0025 M parachloromercuribenzoate was recovered with 0.04 M cysteine. The molecule deteriorated while attempts were made to obtain physical chemical data; consequently, the molecular weight was calculated from an amino acid analysis and found to be 98,800. The reliability of this value is problematical. Human plasma was analyzed and found to contain 13 U./ml Ac-globulin. After 4 days storage, at room temperature, the prolonged prothrombin time of that plasma was completely restored with 13 units of Ac-globulin, which is equivalent to 8 μg.

scholarly journals THE MOLECULAR WEIGHT OF ANTIBODIES

1937 ◽  
Vol 65 (3) ◽  
pp. 393-414 ◽  
Author(s):  
Michael Heidelberger ◽  
Kai O. Pedersen
Keyword(s):  
Molecular Weight ◽  
Molecular Species ◽  
Principal Component ◽  
The Other ◽  
Type I ◽  
Egg Albumin ◽  
Type Iii ◽  
Sedimentation Constant ◽  
Other Hand ◽  
Immune Rabbit

1. Highly purified rabbit Type III pneumococcus anticarbohydrate proved to be homogeneous in the ultracentrifuge and its sedimentation constant, 7.0·10–13, did not differ from that of the principal component of normal rabbit globulin or of immune rabbit globulin containing up to 50 per cent of anti-egg albumin. The molecular weight of antibody in the rabbit is therefore probably very close to that of the principal normal globulin component, namely, 150,000. 2. Highly purified horse Type I pneumococcus anticarbohydrate, on the other hand, was only homogeneous in the ultracentrifuge when prepared from sera stored without preservative. Its sedimentation constant, 18.4·10–13, coincided with that of the principal globulin component in most of the Felton solutions and purified antibody solutions studied. The molecular weight of pneumococcus anticarbohydrate in the horse is probably three to four times that of the principal normal globulin component. 3. The significance of the differences between pneumococcus anticarbohydrate formed in the rabbit and in the horse is discussed. 4. Results are given of ultracentrifuge studies on the molecular species in solutions of egg albumin-anti-egg albumin specific precipitates dissolved in excess egg albumin. The implications of the results are discussed.

PROPERTIES OF BOVINE Ac-GLOBULIN CONCENTRATES AND METHODS OF PREPARATION

1963 ◽  
Vol 41 (12) ◽  
pp. 2409-2421 ◽  
Author(s):  
Nobuo Aoki ◽  
Charles R. Harmison ◽  
Walter H. Seegers
Keyword(s):  
Human Plasma ◽  
Ammonium Sulphate ◽  
Protein Concentration ◽  
Room Temperature ◽  
Specific Activity ◽  
Barium Carbonate ◽  
Dry Weight ◽  
Physical Chemical ◽  
Bovine Plasma ◽  
Refrigerator Temperature

A procedure is described for retaining bovine plasma Ac-globulin activity as one part of the protein from plasma for every 1000 parts removed. The yields averaged 15%. The procedure involves removal of prothrombin with barium carbonate, isoelectric fractionation, fractionation with ammonium sulphate, chromatography on Amberlite IRC-50, and a second fractionation with ammonium sulphate. The procedure requires 2 days; however, the first day completes up to chromatography and the concentrate at that time is quite useful for many purposes. It is more stable than the product obtained after chromatography and the yields are higher. In absence of salts Ac-globulin is quite insoluble at pH 5.0. The final product usually contained some impurity. With the analytical ultra-centrifuge the S20in 0.1 M potassium chloride solution was found to be 4.2 at a protein concentration of 12.4 mg/ml. The specific activity was 1500 U./mg dry weight. Bovine plasma contains 120 U./ml or about 9 mg/100 ml. Assuming the same specific activity for human plasma the concentration is most likely near 1 mg/100 ml. The best stability conditions found were: 50% glycerol, pH 7.0, and 0.1 M calcium chloride. Under those conditions at room temperature all activity was retained 6 to 7 hours, at refrigerator temperature 24 hours, and at −60 °C for 1 month. In rabbits, antibodies were readily produced. Oxidizing agents destroyed the activity, while reducing agents did not, nor did they tend to stabilize. SH blocking agents destroyed the activity. The loss of activity in the presence of 0.0025 M parachloromercuribenzoate was recovered with 0.04 M cysteine. The molecule deteriorated while attempts were made to obtain physical chemical data; consequently, the molecular weight was calculated from an amino acid analysis and found to be 98,800. The reliability of this value is problematical. Human plasma was analyzed and found to contain 13 U./ml Ac-globulin. After 4 days storage, at room temperature, the prolonged prothrombin time of that plasma was completely restored with 13 units of Ac-globulin, which is equivalent to 8 μg.

Ultracentrifugation of Acetylated Thrombin

1960 ◽  
Vol 04 (03) ◽  
pp. 299-302
Author(s):  
Walter H. Seegers ◽  
Robert S. Shepard ◽  
Ricardo H. Landaburu
Keyword(s):  
Potassium Chloride ◽  
Specific Activity ◽  
Dry Weight ◽  
Bovine Thrombin ◽  
Sedimentation Constant

SummaryAcetylated bovine thrombin with a specific activity of 5930 units per milligram dry weight was found to be homogeneous by ultracentrifuge analysis. The sedimentation constant in units of 10−13 sec. at 20° C, in 0.1 M potassium chloride was 3.3. This constant is not concentration dependent.

scholarly journals Pyrene biodegradation capability of two different microalgal strains

2018 ◽  
Keyword(s):  
Molecular Weight ◽  
Aromatic Hydrocarbons ◽  
Biological Treatment ◽  
Growth Parameters ◽  
Dry Weight ◽  
The Other ◽  
Total Chlorophyll ◽  
Incubation Condition ◽  
Chlorella Sp ◽  
Polycyclic Aromatic

<p>Biological treatment is one of the most preferable strategies to complete and safe removal of pollutants. Pyrene (PY) is the first member of high molecular weight polycyclic aromatic hydrocarbons (HMW-PAHs) that represents various concerns to biological life and human health. In this study, two different algal strains exhibited different capability to degrade PY along 30 days of incubation in the light. Dry weight, total chlorophyll, and carotenoids were growth parameters that were determined to detect robust of two algal strains to get used of PY as a source of carbon. Oscillatoria sp. could degrade 95% while Chlorella sp. could degrade 78.71 % of PY (50 mg/L) after 30 days of incubation. Both algal strains could completely remove 10 and 30 mg/L of PY. On the other hand, the degradation capability of Oscillatoria sp. was significantly exceeded than Chlorella sp. under the same incubation condition and at (50 and 100 mg/L) of PY.</p>

A Collaborative Study to Establish the 5th International Standard for Unfractionated Heparin

2000 ◽  
Vol 84 (12) ◽  
pp. 1017-1022 ◽  
Author(s):  
A. Walker ◽  
Barbara Mulloy ◽  
Trevor Barrowcliffe ◽  
Elaine Gray
Keyword(s):  
Molecular Weight ◽  
Unfractionated Heparin ◽  
Specific Activity ◽  
Collaborative Study ◽  
World Health Organisation ◽  
The Other ◽  
World Health ◽  
Expert Committee ◽  
International Standard ◽  
The World

SummaryTwenty-four laboratories participated in a collaborative study to calibrate a replacement for the 4th International Standard for Unfractionated Heparin (82/502). Both candidate materials A and B, gave excellent intra- and inter-laboratory variations (majority of mean %gcv <10%) when assayed against the 4th International Standard. No major differences of potency estimates were found between methods, although the USP method generally gave lower potencies than the other methods and candidate B gave a greater variation between methods than A. Overall, this study showed that the differences between the candidates are marginal. Based on its narrower molecular weight profile, higher specific activity and slightly lower inter-method variation, candidate A, 97/578, was proposed and accepted in October, 1998, by the Expert Committee on Biological Standardisation of the World Health Organisation to be the 5th International Standard for Unfractionated Heparin with an assigned potency of 2031 IU/ampoule.

Cartilage fucoproteins with sites for cross-linking by transglutaminase

1987 ◽  
Vol 65 (4) ◽  
pp. 280-285 ◽  
Author(s):  
J. Michael Bowness
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
High Performance ◽  
Specific Activity ◽  
Weight Fraction ◽  
Dry Weight ◽  
Insoluble Fraction ◽  
Low Molecular Weight ◽  
High Molecular Weight Fraction ◽  
Collagen Iii

Slices of various types of cartilage were incubated with either L-[6-3H]fucose or [1,4-3H(N)]putrescine. Homogenization of the slices and fractionation of the homogenates showed for both labels that an insoluble collagenase-resistant fraction had the highest specific activity (dpm/mg dry weight). Examination of an exhaustive proteolytic digest of this insoluble fraction by ion-exchange high performance liquid chromatography showed the presence of γ-glutamyl[3H]putrescine. Chromatography of solubilized [3H]fucoprotein fractions showed the presence of several low molecular weight peaks, as well as high molecular weight material. Incubation of [3H]fucoprotein extracts with transglutaminase increased the high molecular weight peaks and decreased the low molecular weight ones. Incubation of the cartilage slices with L-[3H]fucose plus 0.5 mM dansylcadaverine, an inhibitor of transglutaminase, caused a decrease in the insoluble and high molecular weight fraction relative to the low molecular weight peaks. It is hypothesized that this is due to inhibition of cross-link formation between fucoprotein components of the cartilage which are transglutaminase substrates. One major low molecular weight peak, which labels with both fucose and putrescine, corresponds in size with the 15 000 subunit of collagen III aminopropeptide, which is known to be a substrate for transglutaminase.

Inhibition of Endocronartium harknesii spore germination by metabolites of Scytalidium uredinicola and S. album, and the influence of growth medium on inhibitor production

1983 ◽  
Vol 61 (8) ◽  
pp. 2147-2152 ◽  
Author(s):  
N. Fairbairn ◽  
M. A. Pickard ◽  
Y. Hiratsuka
Keyword(s):  
Molecular Weight ◽  
Growth Medium ◽  
Spore Germination ◽  
Phosphate Buffer ◽  
Room Temperature ◽  
Malt Extract ◽  
Type Strains

Endocronartium harknesii spores, stored desiccated at −20 °C for 6 weeks, germinated within 6 h at room temperature in phosphate buffer, pH 6. Type strains of Scytalidium album Beyer and Klingström and S. uredinicola Kuhlman et al. and two other isolates of S. uredinicola were compared for their ability to produce compounds which inhibit the germination of E. harknesii spores. Of five media tested, malt extract broth produced best results. Scytalidium album produced a fraction, molecular weight greater than 10 000, which caused spore lysis. Two strains of S. uredinicola formed chloroform extractable material, molecular weight less than 10 000, that inhibited germination of E. harknesii spores.

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