Conformation of immunoglobulin M. I. Characterization of anti-epsilon-1-dimethylamino-5-naphthalenesulfonyl-L-lysine immunoglobulin M antibodies from horse, pig, and shark.
IgM antibodies specific for the fluorophore epsiolon-1-dimethylamino-5-naphthalenesulfonyl-L-lysine(DNS-lysine) were elicited in the horse and nurse shark by immunization with a DNS-lysine streptococcal conjugate; the antibodies were purified by specific adsorption with an immunoadsorbent followed by gel filtration to select the IgM class (molecular weight 900 000). About 90% of the equine anti-DNS was IgM.DNS-Lysine, when bound in the combining sites of a population of these anti-DNS IgM antigodies from horse and nurse shark, as well as from pig, exhibited a marked fluorescence enhancement and shift of the emission spectrum to shorter wavelengths compared with emission in aqueous solution; these results indicate that the environments of the anti-DNS combining sites of  this population were relatively hydrophovic. Approximately one-third of the ten possible combining sites in each of these anti-DNS IgM species bound DNS-lysine in this manner with an average intrinsic association constant (Ko) of greater than  10(6) M-1. Small differences were noted in binding behavior among the three species of antibodies. The enzymatic susceptibility of equine IgM was similar to that of human IgM. (Fab')2mu, Fab'mu, and Fabmu fragments were prepared following digestion with pepsin. These fragments could be clearly differentiated on the basis of molecular size. They bound DNS-lysine with the same affinity as intact IgM  and the DNS-lysine-fragment  complexes exhibited the same spectral properties as the parent IgM. It was concluded that the anti-dNs IgM antibodies from all three species, as well as the enzymatic fragments, were suitable for nanosecond depolarization studies which are reported in the accompanying paper Holowka, D.A., and Cathou, R.E. (1976), Biochemistry, the following papter in this issue.
D000818:Animals; D000906:Antibodies / Q000302:isolation & purification; D001428:Bacterial Vaccines; D001666:Binding Sites, Antibody; D003619:Dansyl Compounds / Q000276:immunology*; D006736:Horses; D007075:Immunoglobulin M / Q000032:analysis* / Q000302:isolation & purification; D008239:Lysine / Q000276:immunology*; D008970:Molecular Weight; D010446:Peptide Fragments / Q000032:analysis; D012754:Sharks; D013050:Spectrometry, Fluorescence; D013291:Streptococcus / Q000276:immunology; D013552:Swine
D000906:Antibodies; D001428:Bacterial Vaccines; D003619:Dansyl Compounds; D007075:Immunoglobulin M; D010446:Peptide Fragments; D008239:Lysine