The Effect Of Thrombin On The Electrophoretic Mobility And Functional Activity Of Various Factor VIII Molecular Forms

1981 ◽  
Author(s):  
M J Seghatchian ◽  
I I Mackie
Keyword(s):  
Molecular Weight ◽  
Factor Viii ◽  
Conformational Changes ◽  
Functional Activity ◽  
Gel Electrophoresis ◽  
Molecular Forms ◽  
Agarose Gel ◽  
Dependent Manner ◽  
Agarose Gel Electrophoresis ◽  
Concentration Dependent Manner

Several distinct populations of factor VIII procoagulant activity (VIIIC) were observed in some clinical concentrates after agarose gel electrophoresis. It is not yet known whether these different forms are the products of proteolytic digestion, or subtle conformational changes of FVIII structure, which may occur during concentrate preparation. Low thrombin concentrations activate FVIII in a time/concentration dependent manner, and the 2 proteins have high affinities for one another. We have studied the properties of the various FVIII forms before and after incubation of concentrate with highly purified α-thrombin. Agarose gel electrophoresis was used to monitor: a) quantitative changes in functional activity (by 2 stage clotting (VIII:C) and amidolytic (VIII: CAm) assays); b) the binding of 125I- anti VIII:C (*IgG) to FVIII: C; c) the crossed immunoelec- trophoretic (CIE) pattern of FVIII: RAg.In the absence of thrombin, FVIII concentrate showed 2 main populations of FVIII: C and VIII: CAm. The radioactivity (VIII: CAg) pattern correlated with the slower moving of these populations. Low thrombin concentrations (0.0001-0.01 u/nl) caused no detectable change in FVIII: C level in the slow peak but additional peaks of activity appeared at the sample well, and between the well and slow peak. This was accompanied by a decrease in the fast moving peak, and the appearance of a third new peak with slightly less mobility. Higher thrombin concentrations (0.5u/ml) caused a marked decrease in the slow moving peak, with a concomitant increase in the well peak, by all methods. Increasing thrombin concentration progressively reduced *IgG binding, and a pattern similar to serum was obtained. Thrombin caused a slightly faster migration of FVIII: RAg, a well peak was sometimes seen. Thrombin therefore appeared to act preferentially on the lower molecular weight FVIII activity, and the high molecular weight peaks that occurred may represent complexes of FVIII with thrombin and/or other contaminant proteins present in concentrate such as fibrinogen or fibronectin.

scholarly journals Plasmid studies ofSalmonella typhimuriumphage type 179 resistant to ampicillin, tetracycline, sulphonamides and trimethoprim

1980 ◽  
Vol 85 (2) ◽  
pp. 293-300 ◽  
Author(s):  
D. M. Anderson
Keyword(s):  
Escherichia Coli ◽  
Molecular Weight ◽  
New Zealand ◽  
Salmonella Typhimurium ◽  
National Health ◽  
Gel Electrophoresis ◽  
Phage Type ◽  
Agarose Gel ◽  
Agarose Gel Electrophoresis ◽  
K 12

SUMMARYSixteen strains ofSalmonella typhimuriumphage type 179 were referred to the National Health Institute, Wellington, New Zealand, from 1977 to 1979. This phage type had not been observed here before 1977. All strains were resistant to ampicillin, several were also resistant to tetracycline, and several were resistant to ampicillin, tetracycline, sulphafurazele and trimethoprim. All resistances could be transferred toEscherichia coliK 12. Plasmids from these strains and their transconjugants were characterized by agarose gel electrophoresis. It appears that resistance to sulphafurazole and trimethoprim is carried on a plasmid with a molecular weight of 5·2 Mdal and that resistance to ampicillin and tetracycline is carried on a plasmid with a molecular weight of approximately 60 Mdal.

scholarly journals Purification of human C4b-binding protein and formation of its complex with vitamin K-dependent protein S

1983 ◽  
Vol 209 (3) ◽  
pp. 847-856 ◽  
Author(s):  
B Dahlbäck
Keyword(s):  
Molecular Weight ◽  
Complex Formation ◽  
Vitamin K ◽  
Gel Electrophoresis ◽  
Binding Protein ◽  
Protein S ◽  
Agarose Gel ◽  
Agarose Gel Electrophoresis ◽  
Dependent Protein ◽  
Molecular Weight Form

C4b-binding protein was purified from human plasma in high yield by a simple procedure involving barium citrate adsorption and two subsequent chromatographic steps. Approx. 80% of plasma C4b-binding protein was adsorbed on the barium citrate, presumably because of its complex-formation with vitamin K-dependent protein S. The purified C4b-binding protein had a molecular weight of 570 000, as determined by ultracentrifugation, and was composed of about eight subunits (Mr approx. 70 000). Uncomplexed plasma C4b-binding protein was purified from the supernatant after barium citrate adsorption. On sodium dodecyl sulphate/polyacrylamide-gel electrophoresis in non-reducing conditions and on agarose-gel electrophoresis it appeared as a doublet, indicating two forms differing slightly from each other in molecular weight and net charge. The protein band with the higher molecular weight in the doublet corresponded to the C4b-binding protein purified from the barium citrate eluate. Complex-formation between protein S and C4b-binding protein was studied in plasma, and in a system with purified components, by an agarose-gel electrophoresis technique. Protein S was found to form a 1:1 complex with the higher-molecular-weight form of C4b-binding protein, whereas the lower-molecular-weight form of C4b-binding protein did not bind protein S. The KD for the C4b-binding protein-protein S interaction in a system with purified components was approx. 0.9×10(-7) M. Rates of association and dissociation at 37 degrees C were low, namely about 1×10(3) M-1 . S-1 and 1.8×10(-4)-4.5×10(-4) S-1 respectively. In human plasma free protein S and free higher-molecular-weight C4b-binding protein were in equilibrium with the C4b-binding protein-protein S complex. Approx. 40% of both proteins existed as free proteins. From equilibrium data in plasma a KD of about 0.7×10(-7) M was calculated for the C4b-binding protein-protein S interaction.

Notizen: The Structural Components of Hog Cholera Virus

1977 ◽  
Vol 32 (5-6) ◽  
pp. 456-458 ◽  
Author(s):  
P.-J. Enzmann ◽  
Hilde Rehberg
Keyword(s):  
Molecular Weight ◽  
Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Structural Proteins ◽  
Agarose Gel ◽  
Agarose Gel Electrophoresis ◽  
Structural Components ◽  
Molecular Weights ◽  
Hog Cholera ◽  
Hog Cholera Virus

Abstract Hog cholera virus grown in PK-15 cells and SK-cells was labeled with [35S] methionine and [3H] uridine. At least 3 polypeptides were resolved by polyacrylamide gel electro­ phoresis after disruption of the virus with sodium dodecyl-sulfate. The molecular weights of the structural proteins were determined to be 55000 (p55), 46000 (gp46), and 36000 (p36). The molecular weight of the viral RNA was determined to be about 4 X 106 in polyacrylamide-agarose-gel electrophoresis. In sucrose gradients the RNA has a S20,w value of 40 -45S.

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