Purification of factor VIII/von Willebrand factor from human plasma on immobilized lentil lectin.
Human factor VIII/von Willebrand factor (FVIII/vWF) was shown to bind to immobilized lectins from Arachis, Ulex, Concanavalia, and Lens species. The protein/lectin interaction displayed higher affinities for the lectins from the last two species. However, the Lens culinaris lectin immobilized on Sepharose 4B (LCA-Sepharose) provided a more selective and flexible affinity system for the purification of FVIII/vWF than Concanavalia lectin. Chromatography on LCA-Sepharose of a purified FVIII containing a small proportion of vWF required a weak acidic medium (pH 6.3) and relatively slow kinetics (about 20 cm/h flow rate). The bound FVIII was specifically dissociated from LCA-Sepharose by methyl-alpha-D-mannopyranoside, and to a lesser extent by other monosaccharides such as D-glucose, methyl-alpha-D-glucopyranoside, D-mannose, and D-galactose. Application to whole plasma resulted in a capacity for FVIII/vWF of about 28 U/ml gel. Specific activities for eluted FVIII and vWF were 3 and 2.2 IU/mg protein, respectively, with respective FVIII:c and vWF:RCo recoveries of 57 and 40% from starting plasma. Coagulation factors II, X, VII, IX, V, and XI and fibrinogen were eliminated in the LCA matrix breakthrough fraction, improving the stability of the purified FVIII molecule. Purity of the LCA eluate was further enhanced by ion-exchange chromatography on DEAE-Fractogel TSK 650 M which reduced the amount of protein contaminants and provided a FVIII/vWF fraction with higher specific activity (45-80 IU/mg protein depending on the chromatographic conditions). The overall process yield was 45 and 25% for FVIII:c and vWF:RCo, respectively.
Zhou
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FL
Centre Régional de Transfusion Sanguine de Lille, France.
Burnouf-Radosevich
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Burnouf
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D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D005169:Factor VIII / Q000302:isolation & purification*; D006801:Humans; D037102:Lectins; D009005:Monosaccharides; D037121:Plant Lectins; D010949:Plasma / Q000737:chemistry*; D011485:Protein Binding; D012685:Sepharose; D014841:von Willebrand Factor / Q000302:isolation & purification*
D037102:Lectins; D009005:Monosaccharides; D037121:Plant Lectins; C033619:lentil lectin; D014841:von Willebrand Factor; D005169:Factor VIII; D012685:Sepharose