1. A series of Dnp (dinitrophenyl) nitroxide spin labels was used to map the dimensions of the combining site of the Dnp-binding immunoglobulin A myeloma protein MOPC 315. The method compares the observed e.s.r. (electron-spin-resonance) hyperfine splittings with those calculated on the basis of different postulated motions for the spin label. The analysis is complicated by the sensitivity of the e.s.r. hyperfine splitting to the overall ‘tumbling’ time of the antibody–hapten complex and the polarity of the spin-label's environment. When these effects are considered quantitatively, it is then possible to determine the degree of mobility of each hapten which is allowed by the shape of the combining site. 2. The dinitrophenyl ring is rigidly held, and the depth of the site is 1.1–1.2nm and has lateral dimensions at the entrance to the site ≥0.6nm×0.9nm. The analysis of the results for spin-labelled haptens with chiral centres allows these lateral dimensions to be refined to 0.8nm and 1.1nm, and it is shown that the site is asymmetric with respect to the plane of the dinitrophenyl ring. 3. A polarity profile of the combining site was also obtained and a positively charged amino acid residue, possibly arginine-95L (light chain), was located at the entrance to the site. 4. The binding of Gd(III) to the antibody–hapten complexes results in quenching of the e.s.r. signal of the nitroxide. By using La(III) as a control, the paramagnetic contribution to the quenching is measured. 5. Analysis of the differential quenchings of the enantiomers of two five-membered nitroxide ring spin labels gives two possible locations of the metal-binding site. One of these is equidistant (0.7nm) from each of the three dinitrophenyl aromatic protons, and nuclear-magnetic-resonance relaxation studies, at 270MHz, on solutions of dinitrobenzene, Gd(III) and the Fv fragment (variable region of heavy and light chain) from protein MOPC 315 support this location for the metal site. 6. The e.s.r. and metal-binding data were then compared with the results of a model of the combining site constructed on the basis of framework invariance in immunoglobulins [Padlan, Davies, Pecht, Givol & Wright (1976) Cold Spring Harbor Symp. Quant. Biol.41, in the press]. The overall agreement is very good. Assignments of possible chelating groups for the metal can be made.
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August 1977
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Research Article|
August 01 1977
The gross architecture of an antibody-combining site as determined by spin-label mapping
Brian J. Sutton;
Brian J. Sutton
*Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, U.K.
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Peter Gettins;
Peter Gettins
*Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, U.K.
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David Givol;
David Givol
†Department of Chemical Immunology, Weizmann Institute of Science, Rehovot, Israel
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Derek Marsh;
Derek Marsh
*Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, U.K.
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Simon Wain-Hobson;
Simon Wain-Hobson
*Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, U.K.
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Keith J. Willan;
Keith J. Willan
*Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, U.K.
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Raymond A. Dwek
Raymond A. Dwek
*Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, U.K.
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Publisher: Portland Press Ltd
Online ISSN: 1470-8728
Print ISSN: 0264-6021
© 1977 London: The Biochemical Society
1977
Biochem J (1977) 165 (2): 177–197.
Citation
Brian J. Sutton, Peter Gettins, David Givol, Derek Marsh, Simon Wain-Hobson, Keith J. Willan, Raymond A. Dwek; The gross architecture of an antibody-combining site as determined by spin-label mapping. Biochem J 1 August 1977; 165 (2): 177–197. doi: https://doi.org/10.1042/bj1650177
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