The subunit structure of normal and hemophilic factor VIII

G. A. Shapiro, J. C. Andersen, S. V. Pizzo, P. A. McKee

Research output: Contribution to journalArticle

79 Scopus citations

Abstract

Human factor VIII from normals and hemophiliacs was partially purified by ethanol and polyethylene glycol precipitations. Final purification was achieved by gel filtration on 2 or 4% agarose or ion exchange chromatography on diethylaminoethyl cellulose. Comparable amounts of highly purified protein were obtained from normal and hemophilic plasma following the agarose chromatography step. Highly purified factor VIII was not dissociated by 6 M guanidine hydrochloride or 1% sodium dodecyl sulfate. However, when reduced by β mercaptoethanol and analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis, a single subunit species with an estimated 195,000 molecular weight was found for both normal and hemophilic factor VIII. By sedimentation equilibrium analysis, the normal factor VIII subunit was homogeneous and had an estimated molecular weight of 202,000. The subunit polypeptides from normal or hemophilic factor VIII contained carbohydrate. Each was homogeneous by isoelectric focusing. Immunodiffusion of purified normal and hemophilic factor VIII against rabbit antiserum to purified normal human factor VIII showed a single line of precipitation. Very low concentrations of purified human thrombin initially increased the activity of normal factor VIII about threefold and then progressively destroyed activity by 3 hr. Only minimal activation occurred with hemophilic factor VIII. Both the activation and inactivation of normal and hemophilic factor VIII were unaccompanied by detectable changes in subunit molecular weight. These findings may have implications for the definition of the molecular defect in hemophilic factor VIII.

Original languageEnglish (US)
Pages (from-to)2198-2210
Number of pages13
JournalJournal of Clinical Investigation
Volume52
Issue number9
DOIs
StatePublished - 1973

ASJC Scopus subject areas

  • Medicine(all)

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