A general approach to the localization of antigenic determinants of a linear type in proteins of unknown primary structure.
A method is proposed which permits the localization of antigenic determinants of a linear type on the polypeptide chain of a protein molecule of unknown primary structure. An antigen modified with maleic anhydride at the amino-terminal groups and at the epsilon-NH2 groups of lysine residues was subjected to partial enzymic digestion, so that the antigenic protein had, on average, less than one cleavage site per polypeptide chain. The resultant ends were labeled with 125I-labeled Bolton and Hunter reagent and the maleic group removed. The detection of the two larger labeled fragments (a longer one which still could bind to a monoclonal antibody and a shorter one which was incapable of binding) made it possible to determine the distance from the antigenic determinant to the C-terminus of the polypeptide chain. The position of the antigenic determinant could be established in more detail using partial chemical degradation of the original antigen using information about the maximal length of a fragment which has lost its ability to interact with the monoclonal antibody. The method has been applied to bovine tryptophanyl-tRNA synthetase (EC 6.1.1.2).
Beresten
S F
SF
Institute of Molecular Biology, U.S.S.R. Academy of Sciences, Moscow.
Rubikaite
B I
BI
Kisselev
L L
LL
D000595:Amino Acid Sequence; D000604:Amino Acyl-tRNA Synthetases / Q000276:immunology*; D000818:Animals; D000911:Antibodies, Monoclonal; D000937:Antigen-Antibody Reactions; D002417:Cattle; D003488:Cyanogen Bromide; D000939:Epitopes / Q000302:isolation & purification*; D015151:Immunoblotting; D007163:Immunosorbent Techniques; D008970:Molecular Weight; D010446:Peptide Fragments / Q000276:immunology; D010449:Peptide Mapping / Q000379:methods; D010455:Peptides / Q000276:immunology*; D014369:Tryptophan-tRNA Ligase / Q000276:immunology*
D000911:Antibodies, Monoclonal; D000939:Epitopes; D010446:Peptide Fragments; D010455:Peptides; D000604:Amino Acyl-tRNA Synthetases; D014369:Tryptophan-tRNA Ligase; D003488:Cyanogen Bromide