Effect of detergents on the structure of integral membrane proteins of Sendai virus studied with size-exclusion high-performance liquid chromatography and monoclonal antibodies.
The integral membrane proteins of Sendai virus, the fusion protein F (Mr = 65,000) and the haemagglutinin-neuraminidase protein HN (Mr = 68,000), were used as a model protein mixture. They were subjected to size-exclusion high-performance liquid chromatography on Superose 6HR columns with eluents containing various additives in order to solubilize the proteins. The effect of the additives on the structure of the membrane proteins was investigated with conformation-dependent monoclonal antibodies, either directed against F or HN protein, and by determination of the haemagglutinating capacity of the HN protein. The results show that the structure of the HN protein is more easily disturbed by eluents than that of the F protein. When the elution conditions are mild, e.g., 0.1% octylglucoside, the structure of both proteins is conserved but no separation is obtained. Elution with a buffer containing 0.05% sarkosyl (dodecyl methylglycine sodium salt) did not affect the structure and resulted in pure F protein. Pretreatment of the Amberlite XAD-2-treated Sendai virus envelope extract with 4% sodium dodecyl sulphate (SDS) and elution with 0.1% SDS in 50 mM sodium phosphate (pH 6.5) altered the structure of the HN protein but resulted in purification of the tetramer and the dimer of the HN protein, and the monomer of the F protein.
Welling-Wester
S
S
Department of Medical Microbiology, Rijksuniversiteit Groningen, The Netherlands.
Kazemier
B
B
Orvell
C
C
Welling
G W
GW
D000911:Antibodies, Monoclonal; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D003902:Detergents; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D006386:Hemagglutination Tests; D008562:Membrane Glycoproteins / Q000032:analysis; D008565:Membrane Proteins / Q000032:analysis*; D008968:Molecular Conformation; D010222:Parainfluenza Virus 1, Human / Q000032:analysis*; D013501:Surface-Active Agents; D014764:Viral Proteins / Q000032:analysis*
D000911:Antibodies, Monoclonal; D003902:Detergents; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D013501:Surface-Active Agents; D014764:Viral Proteins