Localization of Affinity-Labeled Residues on the Heavy and Light Chain of Two Myeloma Proteins with Anti-Hapten Activity

J. Haimovich, H. N. Eisen, E. Hurwitz, D. Givol

Research output: Contribution to journalArticlepeer-review

Abstract

Two mouse myeloma proteins with anti-2,4-dinitrophenyl (Dnp) activity (proteins-315 and -460) were affinity labeled with bromoacetyl derivatives of Dnp ligands. Bromoacetyl-N6-Dnp-L-lysine labeled a lysyl residue on the heavy chain of protein-315, and bromoacetyl-N1-Dnp-ethyl-enediamine labeled a tyrosyl residue on the light chain of protein-315 and a lysyl residue on the light chain of protein-460. Partial sequencing of the isolated labeled tryptic peptides indicated that lysine-54 and tyrosine-34 were the labeled residues on heavy and light chains, respectively, of protein-315, whereas lysine-54 was the labeled residue on the light chain of protein-460. All of the labeled residues are located within restricted hypervariable segments of the variable regions of their respective chains, suggesting that the specific combining sites of the intact immunoglobulins are formed by amino acid residues of the hypervariable segments. A tryptophan residue was present in each of the affinity-labeled tryptic peptides and the Dnp moiety of the peptides from protein-315 showed a red shift in absorption spectrum, resembling that observed when Dnp ligands are specifically bound noncovalently in the combining sites of the intact protein or in those of conventionally raised anti-Dnp antibodies. The results suggest that in the isolated peptides (and perhaps in the intact protein as well) the Dnp group makes contact with Trp-49 or Trp-37 of heavy and light chain, respectively, of protein-315.

Original languageEnglish
Pages (from-to)2389-2398
Number of pages10
JournalBiochemistry
Volume11
Issue number13
DOIs
StatePublished - 1 Jun 1972
Externally publishedYes

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