Contribution of tryptophan residues to the combining site of a monoclonal anti dinitrophenyl spin-label antibody.
Two Fab fragments of the monoclonal anti dinitrophenyl (DNP) spin-label antibody AN02 were prepared by recombination of specifically deuterated heavy and light chains. In the recombinant H(I)L(II) all the tyrosines and phenylalanines were perdeuterated as were the tryptophan residues of the heavy chain. In the recombinant H(II)L(I) all the tyrosines and phenylalanines were perdeuterated as were the tryptophan residues of the light chain. Saturation of three resonances of H(I)L(II), assigned to tryptophan protons of the light chain, resulted in magnetization transfer to the aromatic proton at position 6 of the DNP ring and to the CH2 protons of the glycines linked to the DNP in a diamagnetic hapten (DNP-DG). Saturation of three resonances of H(II)L(I) assigned to tryptophan protons of the heavy chain resulted in magnetization transfer to the CH2 protons of the glycines in DNP-DG. From the dependence of the magnetization transfer on the irradiation time, the cross relaxation rates between the involved protons were estimated. The inferred distances between these protons of the hapten and certain tryptophan protons are 3-4 A. It is concluded that in the combining site of AN02 there is one tryptophan from the light chain and one tryptophan from the heavy chain that are very near the hapten. When all tyrosines and phenylalanines were perdeuterated and all tryptophan aromatic protons were deuterated except for the protons at positions 2 and 5, titration of the Fab fragments with variable amounts of paramagnetic hapten showed that one proton from the light chain tryptophan is near (less than 7 A) the unpaired electron and that three other protons are significantly closer than 15 A.(ABSTRACT TRUNCATED AT 250 WORDS)
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA / Q000032:analysis; D003903:Deuterium; D004140:Dinitrophenols; D006241:Haptens; D007140:Immunoglobulin Fab Fragments / Q000235:genetics; D007143:Immunoglobulin Heavy Chains / Q000235:genetics; D007147:Immunoglobulin Light Chains / Q000235:genetics; D009682:Magnetic Resonance Spectroscopy; D011487:Protein Conformation; D011865:Radioisotope Dilution Technique; D014364:Tryptophan
D000911:Antibodies, Monoclonal; D004140:Dinitrophenols; D006241:Haptens; D007140:Immunoglobulin Fab Fragments; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D014364:Tryptophan; D004247:DNA; D003903:Deuterium