Preparation of the Fv fragment from a short-chain mouse IgG2a anti-dansyl monoclonal antibody and use of selectively deuterated Fv analogues for two-dimensional 1H NMR analyses of the antigen-antibody interactions.
The Fv fragment, a univalent antigen-binding unit with a molecular weight of 25,000, has successfully been prepared in high yield by limited proteolysis with clostripain of a short-chain mouse IgG2a anti-dansyl monoclonal antibody in which the entire CH1 domain is deleted [Igarashi, T., Sato, M., Takio, K., Tanaka, T., Nakanishi, M., & Arata, Y. (1990) Biochemistry 29, 5727-5733]. The Fv fragment obtained is stable at room temperature and retains its full antigen-binding capability. It has been shown that selective deuterium labeling of the Fv fragment, which is half the size of the Fab fragment, provides 1H NMR spectral data at a sufficient resolution for a detailed structural analysis of the antigen-combining site. NOESY spectra of an Fv analogue, in which all aromatic protons except for His C2'-H and Tyr C3',5'-H had been deuterated, were measured in the presence of varying amounts of dansyl-L-lysine. On the basis of the NOESY data obtained, it was possible to assign all the ring proton resonances for the dansyl group that is bound to the Fv fragment. It was also possible to obtain information about His and Tyr residues of the Fv fragment in the absence and presence of the antigen. On the basis of the NMR data obtained, we have shown that at least two Tyr residues along with one of the amide groups are directly involved in antigen binding. The mode of interaction of the dansyl ring with these residues in the Fv fragment has briefly been discussed.
Takahashi
H
H
Faculty of Pharmaceutical Sciences, University of Tokyo, Japan.
Igarashi
T
T
Shimada
I
I
Arata
Y
Y
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000936:Antigen-Antibody Complex; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D006859:Hydrogen; D007074:Immunoglobulin G / Q000276:immunology*; D007143:Immunoglobulin Heavy Chains / Q000276:immunology* / Q000302:isolation & purification; D007147:Immunoglobulin Light Chains / Q000276:immunology* / Q000302:isolation & purification; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D051379:Mice; D011487:Protein Conformation
D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D007074:Immunoglobulin G; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D006859:Hydrogen