First determination of the secondary structure of purified factor VIII light chain.
The first analysis of the secondary structure of human factor VIII light chain was performed by c.d. spectroscopy. The purification process described in this paper allowed us to obtain the large amounts of purified factor VIII light chains required for c.d. experiments. Since this 80 kDa protein is non-covalently associated with a heavy chain to form the active molecule, isolated factor VIII light chains were obtained after immunoadsorption and dissociation of the immobilized active complexes by EDTA. Furthermore, factor VIII light chains were discriminated from the residual active complexes and the free heavy chains by a final ion-exchange-chromatography step. This f.p.l.c. analysis showed that factor VIII light chains were less electronegative than the active complexes. The results of conformational analysis by c.d. show that the protein possesses a high degree of regular secondary structure (58%) with approx. 22% of alpha-helix and 36% of beta-strand structures. The protein was completely unfolded by 3 M-guanidine hydrochloride. The results obtained from the analysis of c.d. spectra were compared with those predicted from three different statistical methods based on amino-acid sequence. The secondary structure information obtained from these methods was in good agreement with the c.d. results. These results were comparable with the secondary structure prediction of ceruloplasmin, a protein known to show sequence identity to factor VIII.
Bihoreau
N
N
T.M. Innovation (Centre National de Transfusion Sanguine Institut Mérieux), Les Ulis, France.
Fontaine-Aupart
M P
MP
Lehegarat
A
A
Desmadril
M
M
Yon
J M
JM
D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D002942:Circular Dichroism; D004492:Edetic Acid; D005169:Factor VIII / Q000737:chemistry* / Q000302:isolation & purification; D006801:Humans; D007163:Immunosorbent Techniques; D017433:Protein Structure, Secondary
D005169:Factor VIII; D004492:Edetic Acid