Affinity Labeling of the Active Sites of Rabbit Anti-2,4-dinitrophenyl Antibodies with m-Nitrobenzenediazonium Fluoroborate<sup>*</sup>

Good, A.H.; Traylor, Patricia S.; Singer, S. J.

doi:10.1021/bi00855a031

Cited by 33 publications

(44 citation statements)

References 21 publications

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“…We now report results obtained when MOPC 315 was reacted with diazonium reagents under the conditions used to label rabbit antibodies to dinitrophenyl determinants (7,8).…”

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confidence: 99%

“…The euglobulin (1 to 2 percent solution) was reduced with 0.01M dithiothreitol at room temperature for 1 hour, and the reduced protein was alkylated with iodoacetamide, in 10 percent molar excess over sulfhydril reagent, at pH 8.0 for 15 minutes at room temperature. The reduced alkylated preparation was then applied to a G-200 Sephadex column equilibrated with a pH 8 In all cases, the labeling reactions were conducted on protein solutions having an optical density of 4 at 280 nm. For analytical work, reaction volumes of 0.5 ml were routinely used.…”

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confidence: 99%

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Affinity Site Labeling of a Mouse Myeloma Protein Which Binds Dinitrophenyl Ligands

Metzger

Potter

1968

Science

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A mouse myeloma protein of the immunoglobulin A class, which specifically binds dinitrophenyl ligands, has been successfully affinity labeled (site-directed labeling) with several diazonium reagents, leading to inactivation of the combining sites. The labeling reagents reacted only with tyrosine residues of light chain origin. The 7S subunit of the myeloma protein appears to contain only one reactive site.

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“…We now report results obtained when MOPC 315 was reacted with diazonium reagents under the conditions used to label rabbit antibodies to dinitrophenyl determinants (7,8).…”

mentioning

confidence: 99%

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confidence: 99%

Affinity Site Labeling of a Mouse Myeloma Protein Which Binds Dinitrophenyl Ligands

Metzger

Potter

1968

Science

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“…Materials and Methods.-Rabbit anti-DNP Ab, elicited by immunization with DNPbovine 7-globulin, were purified7 and affinity-labeled with H3-m-nitrobenzenediazonium fluoborate (H3-MNBDF), 8 and the labeled H and L chains were isolated,9 as in our previous studies. Mouse anti-DNP Ab were raised to DNP-keyhole limpet hemocyanin in Swiss-Webster mice in which Ehrlich ascites tumor cells were intraperitoneally implanted.…”

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confidence: 99%

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

Singer¹,

Thorpe²

1968

Proc. Natl. Acad. Sci. U.S.A.

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By the method of affinity-labeling1 it has been possible to attach chemical tags to amino acid residues that, by a number of stringent criteria, are contact residues within the active sites of antibody (Ab) molecules.2' With IgG Ab specific to three different benzenoid haptens from four different mammalian species,3 4 it was found that tyrosine residues on both heavy (H) and light (L) chains were affinity-labeled by the specific diazonium reagents used. These results were interpreted3 to mean that structurally unique and characteristic tyrosine residues were present in all the active sites that were affinity-labeled. To identify these residues, we have recently isolated and characterized dipeptide fragments bearing the affinity label from the H and L chains of rabbit and mouse IgG Ab specific for the 2,4-dinitrophenyl (DNP) group. Our results (1) demonstrate that the labeled residues are indeed unique; (2) establish that the variable segment of the L chain' is directly involved in forming the Ab active site; (3) strongly suggest that the labeled tyrosine is residue 86 from the amino-terminal end of the L chain; and (4) lead to the suggestion that in the Fab fragment (and, in particular, in the active site) of each half of an IgG molecule, the H and L chains are structurally related by a dyad axis of pseudosymmetry.6Materials and Methods.-Rabbit anti-DNP Ab, elicited by immunization with DNPbovine 7-globulin, were purified7 and affinity-labeled with H3-m-nitrobenzenediazonium fluoborate (H3-MNBDF),8 and the labeled H and L chains were isolated,9 as in our previous studies. Mouse anti-DNP Ab were raised to DNP-keyhole limpet hemocyanin in Swiss-Webster mice in which Ehrlich ascites tumor cells were intraperitoneally implanted.'0 The mouse anti-DNP Ab were isolated from the ascitic fluid and affinity-labeled with H3-MNBDF by methods, and with results, analogous to those for rabbit Ab.The labeled chains were digested with Nagarse enzyme (Enzyme Development Corp.) at a weight ratio of enzyme to peptide of 1:50 in 0.2 M NH4HCO3 for 20 hr at 40'C. Gel filtration of the digests was carried out on P-2 Biogel in 0.05 M NH4HCO3, and the elution of radioactivity was monitored. The elution profiles obtained with rabbit-chain digests have been presented elsewhere.6 The profiles for the mouse-chain digests are shown in Figure 1. In all cases, a major fraction of radioactivity, accounting for about 50% of the original affinity label, was obtained. This major fraction, according to gel filtration experiments with calibrated P-2 Biogel columns in phenol: acetic acid: water (1:1:1), was predominantly dipeptide in size (see also below). Very little of the radioactivity was released as free m-nitrobenzeneazotyrosine."' The radioactive peptides from this major fraction were isolated in a pure state and in high yield by taking advantage of the fact that the m-nitrobenzeneazotyrosine-labeled peptides are haptens capable of specific reversible binding to the active sites of anti-DNP Ab. The labeled peptide fraction was mixed with unlabe...

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“…I n some cases the content of azotyrosine was estimated spectrophotometrically after dissolving an aliquot of the peptide in 0.15M NaOH. Molar absorption coefficient 10,020 at 490 nm was used [7].…”

Section: Nonspecific Purification Proceduresmentioning

confidence: 99%

Affinity Labeling by m‐Nitrobenzenediazonium Fluoroborate of Porcine Anti‐Dinitrophenyl Antibodies

Franěk

1973

European Journal of Biochemistry

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From the tryptic digest of y-chains of porcine anti-dinitrophenyl antibody which has been affinity-labeled by m-nitrobenzenediazonium fluoroborate, an azotyrosine-containing peptide consisting of 28 amino acid residues was isolated using affinity chromatography on Sepharosebound antibodies. The peptide contained a half-cystine residue and a tryptophan residue, The thermolysin digest of this peptide yielded several shorter peptides. The amino acid sequence in the vicinity of the half-cystine was revealed by the finding of the peptide Leu-Ser-Cys(0,H). Azotyrosine was found in a nonapeptide, the amino acid sequence of which was characterized by the presence of more than one amino acid in most of the steps of sequential degradation.Comparison of the amino acid sequence in the vicinity of the half-cystine residue and of the partial amino acid sequence of the azotyrosine-containing nonapeptide with the known sequences of heavy chains of man, rabbit, mouse and guinea-pig immunoglobulins showed that the labeled tyrosine of y-chains of porcine anti-dinitrophenyl antibody is located most likely in the hypervariable section between half-cystine 22 and tryptophan 36. The exact determination of the positions of labeled tyrosines in the A-chains of the porcine antibody was facilitated by the fact that the amino acid sequence of the corresponding sections of the polypeptide chains of normal A-chains had been identified before [4,5]. It was the aim of the present work to localize labeled tyrosine in the heavy ( y ) chains of the porcine anti-dinitrophenyl antibody. This task was evidently more difficult than the previous one since there were no data available either on the amino acid sequence of porcine y-chains or on the extent of variability in their variable regions. I n spite of these obstacles it has been found that most of the label is attached to a single site which is not very distant from the first half-cystine residue.

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Affinity Labeling of the Active Sites of Rabbit Anti-2,4-dinitrophenyl Antibodies with m-Nitrobenzenediazonium Fluoroborate^*

Cited by 33 publications

References 21 publications

Affinity Site Labeling of a Mouse Myeloma Protein Which Binds Dinitrophenyl Ligands

Affinity Site Labeling of a Mouse Myeloma Protein Which Binds Dinitrophenyl Ligands

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

Affinity Labeling by m‐Nitrobenzenediazonium Fluoroborate of Porcine Anti‐Dinitrophenyl Antibodies

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Affinity Labeling of the Active Sites of Rabbit Anti-2,4-dinitrophenyl Antibodies with m-Nitrobenzenediazonium Fluoroborate*

Cited by 33 publications

References 21 publications

Affinity Site Labeling of a Mouse Myeloma Protein Which Binds Dinitrophenyl Ligands

Affinity Site Labeling of a Mouse Myeloma Protein Which Binds Dinitrophenyl Ligands

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

Affinity Labeling by m‐Nitrobenzenediazonium Fluoroborate of Porcine Anti‐Dinitrophenyl Antibodies

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Affinity Labeling of the Active Sites of Rabbit Anti-2,4-dinitrophenyl Antibodies with m-Nitrobenzenediazonium Fluoroborate^*