On the location and structure of the active sites of antibody molecules.

Singer, S. J.; Thorpe, Neal O.

doi:10.1073/pnas.60.4.1371

Cited by 32 publications

(5 citation statements)

References 25 publications

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“…Both positions of labeled tyrosines found in the A-chains of porcine anti-dinitrophenyl antibody are in the N-terminal half of the chain which is usually called the variable region. Our results are a t variance with the speculative conclusion by Singer and Thorpe [8,27] that the labeled tyrosine in rabbit and mouse antibodies is in position 86 (corresponding to position 88 in the numbering system used here). Using monoclonal mouse immunoglobulin It is evident that the sections of sequence are homologous.…”

Section: Discussioncontrasting

confidence: 56%

Affinity Labeling by m-Nitrobenzenediazonium Fluoroborate of Porcine Anti-Dinitrophenyl Antibodies. Position of Labeled Tyrosine in the lamba-Chains

Franěk

1971

Eur J Biochem

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The porcine anti-dinitrophenyl antibody was subjected to affinity labeling by m-nitrobenzenediazonium fluoroborate. From the S-sulfo derivative of the labeled antibody light (1 and x ) and heavy chains were isolated. It was found by spectral analysis of the polypeptide chains that the m-nitrobenzenediazonium reagent labeled tyrosine residues. I n the light chains loo/, molecules, in the heavy chains 2i0/,, molecules were labeled. Tryptic digest of labeled 1-chains was resolved by gel chromatography. The label was found to be distributed in two peaks a t a ratio of I : 3. From the labeled peptides of the tryptic digest shorter peptides were prepared by hydrolysis with subtilisin and purified by gel chromatography, paper chromatography and affinity chromatography on Sepharose-bound anti-dinitrophenyl antibodies. Comparison of partial sequences of labeled peptides with known sections of the amino acid sequence of the ].-chains of porcine nonspecific immunoglobulin showed that the main portion of the label is attached to the tyrosine in position 33, a lesser portion to the tyrosine in position 93.Investigation of the ligand binding sites on proteins requires the knowledge of the position of those amino acids in the polypeptide chain which participate in the ligand binding. For this purpose it is necessary to design and synthesize a modified ligand which could form a covalent bond with one or more amino acids constituting the binding site. Such an approach is called affinity labeling as it employs the specific affinity of a ligand for the binding site [ 1,2].For affinity labeling of antibodies it is possible to use a hapten equipped with a reactive group. Metzger and co-workers [3] and Good and co-workers [4] examined several derivatives in the form of diazonium salts to be used for affinity labeling of anti-dinitrophenyl antibodies. They obtained satisfactory results, in particular with m-nitrobenzenediazonium fluoroborate. I n the anti-dinitrophenyl antibodies of both the rabbit and other animal species the reagent was bound t o tyrosine residues [3--51 and the label was found both in the heavy and in the light chains [6].The anti-dinitrophenyl antibodies are heterogeneous with respect to the amino acid sequence. Consequently, the amino acid sequence in the vicinity of the labeled tyrosine is not uniform [7].I n view of this difficulty, Thorpe and Singer [8] determined only the nearest amino acid neighbors of the labeled tyrosines in rabbit and mouse antibodies. Nevertheless, they attempted to draw conclusions about the position of labeled tyrosines in the chains. More favorable conditions for studying labeled peptides were found by Goetzl and Metzger [9,10] who performed affinity labeling of mouse monoclonal immunoglobulin MOPC 315 which binds nitrophenyl ligands. I n this protein only the light chain is labeled by the m-nitrobenzenediazonium reagent. Labeled tyrosine was found in the peptide, the sequence of which yields reliable data for the conclusion that the labeled tyrosine occupies the position 34 [lo]. W...

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Section: Discussioncontrasting

confidence: 56%

Affinity Labeling by m-Nitrobenzenediazonium Fluoroborate of Porcine Anti-Dinitrophenyl Antibodies. Position of Labeled Tyrosine in the lamba-Chains

Franěk

1971

Eur J Biochem

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“…The two interchanges described in the heavy chain aminoterminal sequences might be examples of sustitutions which are not concerned with the structure of the site. Most studies to date indicate that site-related amino acids, i.e., amino acids in the hypervariable regions and amino acids which have been affinity site labeled (24)(25)(26)(27)(28) are not located in the aminoterminal portion of either the light or heavy chain. Substitutions in the aminoterminal sequences of the heavy chain might, however, contribute to the individual antigenic specificity of these proteins.…”

Section: Anti-x44mentioning

confidence: 99%

SIX BALB/c IgA MYELOMA PROTEINS THAT BIND ß-(1 → 6)-D-GALACTAN

Rudikoff

Mushinski

Potter

et al. 1973

The Journal of Experimental Medicine

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Six IgA myeloma proteins of BALB/c origin which bind antigens containing ß-(1 → 6)-D-galactan side chains have been isolated by affinity chromatography on galactoside-BSA-Sepharose columns. Partial amino acid sequences of of the light chains to residue Cys23 and the heavy chains to reside 30 were determined on the automated sequencer. No differences were found among the six VK sequences. Among some 50 partial VK sequences that have thus far been determined these six chains are the only ones thus far identified in this subgroup; at least 25 VK subgroups in the mouse have been identified so far. The heavy chain partial sequences were also very closely related but two differences were found. One protein differed from the other five by having isoleucine instead of leucine at position 5, a second protein differed from the others by having an unidentified amino acid at position 19. Using the highly sensitive inhibition of hemagglutination method it was found that each of the proteins possessed a unique inidividual antigenic determinant.

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“…Helical oligonucleotide antigens were crosslinked to both chains of antibody H241 by UV irradiation of the immune complex (Jang and Stollar, 1990). Both chains of immunization-induced, anti-hapten antibodies are usually affinity-labeled to some extent (Metzger et al, 1964;Press et al, 197 1;Singer and Thorpe, 1968;Weigert et al, 1970). Crystallographic analysis of antibodyhapten and antibody-protein immune complexes usually reveals more antigen contacts with H chain than with L chain CDR residues, but each chain makes a significant number of contacts (Davies et al, 1990;Padlan, 1994).…”

Section: The Roles Of H and L Chain V Regionsmentioning

confidence: 99%