A monoclonal antibody to avidin dissociates quaternary structure and curtails biotin binding to avidin and streptavidin.
An anti-avidin mAb, viz., H12G4, is shown to release bound biotin in a dose-dependent manner from holoavidin and holostreptavidin and inhibit the binding of ligand to the two apoproteins. The release of biotin by this mAb is accompanied by quenching of ligand-induced enhanced fluorescence of the FITC-avidin conjugate. In terms of mechanism of release of bound biotin, we demonstrate that on binding to the Fab fragment of the mAb, the native tetrameric holoavidin undergoes dissociation progressively with time to monomers with no bound biotin associated with the latter. Based on the immunoreactivity associated with defined overlapping fragments of avidin obtained by chemical cleavage, the epitope recognized by mAb H12G4 has been localized to residues 58-96 of the primary sequence. By pepscan method of epitope mapping, this mAb is shown to identify a minimal core sequence of 87RNGK90 in avidin and a corresponding sequence of 84RNAH87 in streptavidin.
Subramanian
N
N
Department of Biochemistry and Centre for Reproductive Biology & Molecular Endocrinology, Indian Institute of Science, Bangalore.
Subramanian
S
S
Karande
A A
AA
Adiga
P R
PR
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000276:immunology*; D001360:Avidin / Q000031:analogs & derivatives / Q000737:chemistry* / Q000276:immunology* / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D001665:Binding Sites / Q000276:immunology; D001710:Biotin / Q000378:metabolism*; D015153:Blotting, Western; D002850:Chromatography, Gel; D003488:Cyanogen Bromide; D004591:Electrophoresis, Polyacrylamide Gel; D018604:Epitope Mapping; D000939:Epitopes / Q000737:chemistry / Q000276:immunology; D016650:Fluorescein-5-isothiocyanate / Q000031:analogs & derivatives; D019811:Hydroxylamine; D006898:Hydroxylamines; D007140:Immunoglobulin Fab Fragments / Q000276:immunology; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D019809:Streptavidin
D000911:Antibodies, Monoclonal; D001426:Bacterial Proteins; D000939:Epitopes; D006898:Hydroxylamines; D007140:Immunoglobulin Fab Fragments; D010446:Peptide Fragments; C036963:fluorescein isothiocyante avidin; D001360:Avidin; D019811:Hydroxylamine; D001710:Biotin; D019809:Streptavidin; D016650:Fluorescein-5-isothiocyanate; D003488:Cyanogen Bromide