Metal identification in human anti-hemophilia A factor (factor VIII)

Anti-hemophilia A factor (FVIII) consists in different heterodimers of heavy and light chains associated by a metallic ion. The integrity of this complex is indispensable for procoagulant activity. Atomic absorption spectrometry with Zeeman effect has been applied to determine the nature of this metal. For this purpose, the different active forms of FVIII were separated by FPLC and characterized by SDS-PAGE. Two peaks were observed, the first corresponding to different FVIII complexes of high molecular mass (ranging from 210-80 kDa to 110-80 kDa) and the second to the heavy-light chain dimer of 90-80 kDa. In all these active fractions, copper atom was identified and a proportionality was measured between the metal concentration and the coagulant activity. Furthermore, the determination of copper and FVIII concentrations indicated that only one copper atom is implicated in the 90-80 kDa association.