Conformation of immunoglobulin M. 2. Nanosecond fluorescence depolarization analysis of segmental flexibility in anti-iε-1-dimethylamino-5-naphthalenesulfonyl-L-lysine anti-immunoglobulin from horse, pig, and shark

Abstract

The rotational motions of immunoglobulin M (IgM) were investigated by the nanosecond fluorescence depolarization technique. The fluorophore .epsilon.-1-dimethylamino-5-naphthalenesulfonyl-L-lysine (DNS-lysine) was specifically bound in the combining sites of anti-DNS IgM antibodies from the horse, pig and nurse shark. Fluorescence lifetime analysis showed the presence of a long lifetime component (21-27 ns) with antibodies from all 3 spp. With the mammalian antibodies, the fluorophore appeared to be rigidly bound in the combining sites as judged by the presence of induced circular dichroism of DNS-lysine (equine antibodies) and single exponential anisotropy decay of the isolated Fab.mu. fragments (equine and porcine antibodies). The small amount of available purified nurse shark antibody did not allow preparation of fragments or induced circular dichroism measurements to directly determine rigidity of fluorophore binding. At least some of the hapten must have been rigidly bound since long rotational correlation times were measured for the shark DNS-lysine-anti-DNS complexes. When the emission anisotropy of the fluorophore-anti-DNS IgM complexes was measured as a function of time, all 3 antibody species exhibited restricted segmental flexibility in the nanosecond time range. When the equine anti-DNS IgM was exposed to 1 M acetic acid for 1 h, the antibody underwent a conformational change which resulted in an increase in its overall flexibility. Comparison of the rotational correlation times of native equine IgM and of proteolytic fragments indicated that flexibility of IgM consists of hindered rotation of the Fab''.mu. segment or a combination of at least 2 modes of motion: rotation of Fab.mu. and/or Fab''.mu. and bending of the entire (Fab'')2.mu. region as a unit. Similar modes of flexibility also occur in native porcine IgM. In acid exposed equine IgM, the major contribution to depolarization is from independent rotation or wagging of the Fab''.mu. segments. Thus, acid apparently causes a conformational change in or near the C[constant region].mu.2 domains. In contrast, flexibility in nurse shark IgM appears to involve only bending of (Fab'')2.mu. as a unit. Apparently segmental flexibility is an essential functional feature of all IgM antibodies and control of this flexibility through domain interactions may play an important role in such conformationally sensitive functions as complement fixation.