ABSTRACT
Heparin is a natural glycosaminoglycan with chains consisting of alternating 1-4 linked residues of sulphated uronic acids (L-iduronic in great proportion) and D- glucosamine attached to a serine-glycine linear protein core. In our previous experiments with a low molecular weight heparin (Mr = 4.000 to 6.000) obtained by partial chemical degradation of the original heparin we could concentrate its anticoagulant activity by precipitation with the first component of the human complement system. In order to confirm these results with a more physiological unfractionated heparin we used commercial heparin from porcine intestinal mucose with a high molecular weight (9.000-15.000) and a specific activity of 179 U/mg. An heparin fraction with high anticoagulant activity was isolated from the precipitate of the interaction between this high molecular weight heparin isolated from a natural source, and the first component of the human complement system. Our results confirmed, in opposition to almost all early literature, that under very strict conditions of pH 6.0, calcium chloride concentration (2 mM), and very low ionic strength (25 mM), the first component of the human complement cascade recognize heparin fractions "enriched" in the high affinity sequence for the antithrombin III.
Subject(s)
Complement C1/metabolism , Heparin/metabolism , Animals , Antithrombin III/metabolism , Calcium/metabolism , Heparin/chemistry , Heparin/isolation & purification , Humans , Hydrogen-Ion Concentration , In Vitro Techniques , Intestinal Mucosa/chemistry , Molecular Weight , SwineABSTRACT
A low molecular weight heparin fraction with high anticoagulant activity was isolated from the precipitate of the interaction with the first component of the human complement system. Our results confirmed the predictions: under very strict conditions of pH (6.3), CaCl2 concentration (2 mM), ionic strength (25 mM) and protein/heparin ratio (1/1), the first component of the complement recognizes the high antithrombin III affinity fraction of heparin, and allows a concentration of the biological activity of the original low molecular weight-heparin.
Subject(s)
Complement C1/metabolism , Heparin, Low-Molecular-Weight/metabolism , Anticoagulants/metabolism , Antithrombin III/metabolism , Complement C1/chemistry , Complement C1/isolation & purification , Heparin, Low-Molecular-Weight/chemistry , Humans , Hydrogen-Ion Concentration , Models, Chemical , Osmolar ConcentrationABSTRACT
A dermatan sulfate (DS) fraction with high anticoagulant activity was isolated from the precipitate of the interaction with Concanavalin A (Con A). Under the optimum conditions found, the interaction depends absolutely on the presence of Ca++ or Mn++ ions. The pH curve of the activity showed an acute maximum at pH 4.4. The active fraction showed, in average 5.4 folds higher specific activity than the fraction that remained in the supernatant. Native DS was desulfated by acid hydrolysis: a drastic reduction in the interaction with Con A was observed during the first ten min. at 100 degrees C and a parallel decrease in the anticoagulant activity was also detected. Sulfate liberation followed the same pattern and after 30 min. at 100 degrees C no sulfate groups remain bound.
Subject(s)
Concanavalin A/metabolism , Dermatan Sulfate/metabolism , Anticoagulants/metabolism , Calcium , Hydrogen-Ion Concentration , MagnesiumABSTRACT
A systematic study of the role of the Ca2+ and Mn2+ ions on the interaction between heparin and Concanavalin A was carried out. The protein was demetallized, and complex formation was followed by a light scattering assay. In the absence of ions no visible interaction was detected. Maximum activity was obtained when Mn2+ ions were added. A similar effect was observed when Ca2+ ions alone were used. Kinetics of the interaction in the presence of optimal Mn2+ and(or) Ca2+ concentrations confirmed the role of Ca2+ in accelerating a conformational change leading to a functional protein. A peculiar effect of activation-inhibition depending on ion concentration was also exhibited by Ca2+. These results confirm that Ca2+ and Mn2+ can occupy both sites on Concanavalin A and activate the protein for binding heparin. They point also to a crucial role of Ca2+ in the binding capacity of the active heparin fraction.
Subject(s)
Blood Coagulation/drug effects , Concanavalin A/pharmacology , Heparin/pharmacology , Binding, Competitive , Calcium/pharmacology , Drug Interactions , Kinetics , Light , Manganese/pharmacology , Models, Biological , Protein Conformation , Scattering, Radiation , SpectrophotometryABSTRACT
Heparin and Concanavalin A complexes were studied under different conditions. Interaction was measured by reading the turbidity at 420 nm. The influence of the pH, heparin, and salt concentration was measured. In the presence of salts pH was very critical, and above pH 5.4 the interaction was practically negligible. At pH 4.6 or 5.2 and the lowest salt concentration compatible with buffering, heparin fractions with different anticoagulant specific activities were detected in the precipitate and in the supernatant, after the interaction. In all cases a significative difference was observed in favor of the heparin isolated from the precipitate. Possibility of an artifact was eliminated by using adequate blanks and running the coagulation tests in the presence of an excess of Concanavalin A.
Subject(s)
Blood Coagulation Tests , Concanavalin A/pharmacology , Heparin/pharmacology , Calibration , Drug Interactions , Fractional Precipitation , Heparin/isolation & purification , Heparin/standards , Hydrogen-Ion Concentration , SaltsABSTRACT
The absence of DNA in the yolk platelets of Bufo arenarum oocytes was verified. Oocytes from the coelomic cavity were used in order to avoid possible contamination by the ovarian tissue. Four methods for DNA analysis were employed, and the possibility of interference was carefully checked. The presence of three possible contaminants, which produced positive reactions from the DNA analysis, was detected. These were the glycosaminoglycan from the superficial layer of the platelets, sucrose, and polyvinylpyrrolidone from the buffer commonly used during the isolation of platelets. The present evidence points to the necessity of using at least two different methods for DNA determination with due regard for their sensitivity and specificity before any firm conclusion can be reached on the presence or absence of DNA in nonnuclear and mitochondrial parts of the oocyte.
Subject(s)
DNA/analysis , Egg Yolk/analysis , Oocytes/analysis , Animals , Bufo arenarum , Cattle , DNA/isolation & purification , Female , Glycosaminoglycans/isolation & purification , Phosphates/analysis , Ribose/analysis , Sucrose/analysis , Thymus Gland , Uronic Acids/analysisABSTRACT
After treatment with D-galactose oxidase to form an aldehyde group, D-galactose or 2-acetamido-2-deoxy-D-galactose reacted with indole-hydrochloric acid to give a colored compound having a spectrum very similar to that of D-galacturonic acid, but with a maximum at 500 nm and a shoulder at 480 nm. The reaction is linear between 16.6 and 83 nmol of sugar per mL of final solution. 2-Amino-2-deoxy-D-galactose gave no reaction, even when 5 mumol were used, and 2-deoxy-D-lyxo-hexose did not interfere either.
