Conformation of immunoglobulin M. 1. Characterization of anti-iε-1-dimethylamino-5-naphthalenesulfonyl-L-lysine immunoglobulin M antibodies from horse, pig, and shark

Abstract

IgM [immunoglobulin M] antibodies specific for the fluorophore .epsilon.-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 (MW 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 antibodies from horse and nurse shark, and 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 hydrophobic. Approximately 1/3 of the 10 possible combining sites in each of these anti-DNS IgM species bound DNS-lysine in this manner with an average intrinsic association constant (K0) > 106 M-1. Small differences were noted in binding behavior among the 3 spp. of antibodies. The enzymatic susceptibility of equine IgM was similar to that of human IgM. (Fab'')2.mu., Fab''.mu. and Fab.mu. 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.