Affinity purification of human tissue factor: interaction of factor VII and tissue factor in detergent micelles.
AUTOR(ES)
Guha, A
RESUMO
Tissue factor, a known initiator of blood coagulation, was found to be active in Triton X-100. A system consisting of tissue factor, factor VIIa, calcium ions, and coagulation factor X generated activated factor X at an appreciable rate. Based on this observation, we coupled human and bovine factor VII to a solid support. Each column bound tissue factor, solubilized in Triton X-100, in a species-specific manner. These interactions required calcium ions; when the columns were washed with detergent containing calcium ions, no tissue factor was eluted. When calcium ions were omitted from the eluant, tissue factor emerged as a sharp peak. Human tissue factor was extracted from an acetone brain powder into 2% Triton X-100. This extract, made 10 mM in CaCl2, was passed over a factor VII column. Human factor VII (1.2 mg) was coupled to 30 ml of Affi-Gel 15. This column bound approximately equal to 15 micrograms of human tissue factor. The eluted material was approximately equal to 25% pure. Final purification was achieved by gel filtration after chymotryptic digestion of contaminants. The tissue factor activity was stable to this treatment. The molecular weight determined by sodium dodecyl sulfate/PAGE (approximately equal to 46,000) was also unchanged by chymotrypsin. The final material was a single band on PAGE, demonstrated similar resistance to tryptic and chymotryptic digestion as bovine tissue factor, and had approximately the same specific coagulant activity as the previously purified bovine material. Tissue factor was also purified from human placenta, yielding a similar protein. A partial 28-residue sequence of the latter has been obtained.
ACESSO AO ARTIGO
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=322845Documentos Relacionados
- The interaction of quinone and detergent with reaction centers of purple bacteria. I. Slow quinone exchange between reaction center micelles and pure detergent micelles.
- Fluorescence resonance energy transfer analysis of lipopolysaccharide in detergent micelles.
- The importance of residues 195-206 of human blood clotting factor VII in the interaction of factor VII with tissue factor.
- Activation of factor VII bound to tissue factor: a key early step in the tissue factor pathway of blood coagulation.
- Staphylococcal alpha-toxin: oligomerization of hydrophilic monomers to form amphiphilic hexamers induced through contact with deoxycholate detergent micelles.