Studies on Binding Sites of Myeloma Proteins

Abstract Mouse IgA myeloma protein S-183 detected as binding the 3-nitro-4-hydroxyl-5-iodophenylacetyl (NIP) groups by precipitation of NIP-protein conjugate, was specifically purified on a NIP-immunoadsorbent (NIP-poly-RSA). The purified protein binds 2 moles NIP-ε-aminocaproate/150,000 g protein with a binding constant (K0) of ~5.5 × 103 M-1. Fab fragments of specifically purified S-183 have one specific NIP-binding site/50,000 g protein, K0 ≈ 3.1 × 103 M-1. The binding sites on S-183 Fab contain arginyl residues, as do those of conventionally induced rabbit anti-NIP antibodies, as shown by loss of binding sites when 63% of S-183 Fab arginine is glyoxalated. Some protection of the sites from glyoxalation is afforded by 0.1 M NIP. Evidence for the degree of specificity of the S-183 sites for NIP obtains from comparison with NIP binding by MOPC-315 mouse IgA myeloma protein (K0 ~2 × 103 M-1) which does not precipitate with NIP-protein and by several human IgG myeloma proteins (K0 ~0.3–0.7 × 103 M-1). MOPC-315 has been subjected to paired-label iodination by Dr. O. A. Roholt (Roswell Park Memorial Institute) in order to isolate a tyrosinecontaining peptide from its specific binding site. Such a peptide has been detected and its isolation and sequencing are under way. Studies on binding of a spin-labeled DNP hapten to MOPC-315 by Dr. L. H. Piette (University of Hawaii) have suggested that the binding site in MOPC-315 is of somewhat different shape (or composition) than the site in rabbit anti-DNP antibody, since the spin-labeled hapten is immobilized to a lesser degree in the former site, as shown by electron spin resonance measurements.