Human factor VIII: purification from commercial factor VIII concentrate, characterization, identification and radiolabeling.
Human factor VIII was purified from commercial factor VIII concentrate with a 12% yield. The specific coagulant activity of purified factor VIII was 8,000 units/mg. In the presence of SDS the purified factor VIII consisted of a variety of polypeptides on polyacrylamide gels, ranging between Mr 80,000 and Mr 208,000. In the absence of SDS the purified factor VIII showed an apparent molecular weight of 270,000 upon Sephadex G200 gel-filtration. The purified factor VIII could be activated by thrombin, which resulted in the disappearance of Mr 108,000-208,000 polypeptides in favor of an Mr 92,000 polypeptide. Treatment with factor Xa also activated factor VIII, whereas treatment with activated protein C resulted in the inactivation of coagulant activity. Coagulant-active 125I-factor VIII was prepared using a lactoperoxidase radioiodination procedure. This 125I-factor had the same characteristics as unlabeled factor VIII. All polypeptides could be precipitated with monoclonal antibodies directed against factor VIII. With 125I-factor VIII a pIapp of 5.7 was found in the presence of urea.
D011232:Chemical Precipitation; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D005169:Factor VIII / Q000302:isolation & purification*; D015944:Factor VIIIa; D006801:Humans; D007120:Immunochemistry; D007457:Iodine Radioisotopes; D007553:Isotope Labeling; D010455:Peptides / Q000302:isolation & purification; D011486:Protein C; D013917:Thrombin / Q000494:pharmacology
D007457:Iodine Radioisotopes; D010455:Peptides; D011486:Protein C; D015944:Factor VIIIa; D005169:Factor VIII; D013917:Thrombin