Folding patterns of immunoglobulin molecules identified by urea gradient electrophoresis.
The reversible denaturant-induced unfolding of immunoglobulin molecules has been analyzed by transverse urea gradient gel electrophoresis and the effects that urea-induced unfolding exerts on the functional properties associated with their variable region, i.e. antigen binding and idiotypic expression, have been determined by Western blot analysis. Results obtained from these experiments indicate that urea-induced unfolding of the immunoglobulin molecule is a highly cooperative reversible process that occurs through a two-state transition with no accumulation of intermediates. The unfolding transition has its midpoint at about 6.5 M urea and appears to be slow on the time scale of electrophoresis. Folding intermediates in rapid equilibrium with the unfolded state as well as molecular forms with different electrophoretic mobility can be detected during refolding reactions. Results from Western blot analysis confirm the highly cooperative reversible urea-induced unfolding of immunoglobulin molecules and demonstrate that the unfolding transition leads to disappearance of both antigen binding and idiotypic expression, whereas the ability to interact with antibodies directed to continuous epitopes of the variable region is preserved. After progressive removal of the denaturing agent, the variable region refolds into structures that regain the functional properties of the native conformation.
Attanasio
R
R
Department of Virology and Immunology, Southwest Foundation for Biomedical Research, San Antonio, Texas 78228-0147.
Stunz
G W
GW
Kennedy
R C
RC
D000818:Animals; D000906:Antibodies / Q000737:chemistry* / Q000302:isolation & purification; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000302:isolation & purification; D015153:Blotting, Western; D015704:CD4 Antigens / Q000276:immunology; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D000939:Epitopes / Q000032:analysis; D015483:HIV Antibodies / Q000737:chemistry / Q000302:isolation & purification; D006801:Humans; D007074:Immunoglobulin G / Q000737:chemistry* / Q000302:isolation & purification; D007145:Immunoglobulin kappa-Chains / Q000737:chemistry* / Q000302:isolation & purification; D007700:Kinetics; D051379:Mice; D017510:Protein Folding; D011817:Rabbits; D014508:Urea / Q000494:pharmacology*
D000906:Antibodies; D000911:Antibodies, Monoclonal; D015704:CD4 Antigens; D000939:Epitopes; D015483:HIV Antibodies; D007074:Immunoglobulin G; D007145:Immunoglobulin kappa-Chains; D014508:Urea