The molecular mechanism of cryoimmunoglobulin precipitation--II. Thermodynamic basis for self-association as determined by fluorescence polarization.
Protein MAT is a homogeneous human IgM (lambda) cryoprecipitating cold agglutinin wherein the Fabmu and Fcmu5 fragment interaction is facilitated at low temps. Thermal dependence of the association of 2,5 DNS labeled Fab MAT with unlabeled Fcmu5 MAT was examined by fluorescence depolarization. The association data, treated by the method of van't Hoff, showed a nonlinear increase in binding with decreased temp, suggestive of certain dynamic changes in the system. Temperature effects on the rotational diffusion of five different 1,5 DNS labeled Fabmu fragments (including two derived from cryoglobulins) were also examined by fluorescence polarization. The linear nature of the Perrin plots derived from the data failed to reveal temp-induced hydrodynamic changes in any of the Fabmu fragments studied. Involvement of carbohydrates in the low-temp self-association of protein MAT was established by the finding that glycopeptides isolated from another IgM molecule (BAZ) could inhibit (as judged by depolarization of fluorescence) the interaction of 2,5 DNS labeled Fabmu MAT and unlabeled Fcmu5 BAZ fragments. These findings indicate that, although cryoprecipitation of protein MAT seemingly involves an antigen-antibody-like reaction between a site on the Fab region and carbohydrate moieties on the Fcmu5 region, no direct evidence for a low-temp-induced conformational change in the Fab region was obtained.
D000373:Agglutinins / Q000276:immunology*; D000937:Antigen-Antibody Reactions; D011232:Chemical Precipitation; D003450:Cryoglobulins / Q000276:immunology*; D005454:Fluorescence Polarization; D005456:Fluorescent Dyes; D006020:Glycopeptides; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000276:immunology; D007141:Immunoglobulin Fc Fragments / Q000276:immunology; D007075:Immunoglobulin M / Q000276:immunology; D011487:Protein Conformation; D013696:Temperature; D013816:Thermodynamics
D000373:Agglutinins; D003450:Cryoglobulins; D005456:Fluorescent Dyes; D006020:Glycopeptides; D007140:Immunoglobulin Fab Fragments; D007141:Immunoglobulin Fc Fragments; D007075:Immunoglobulin M; C029443:cold agglutinins