FOLDING PATTERNS OF IMMUNOGLOBULIN MOLECULES IDENTIFIED BY UREA GRADIENT ELECTROPHORESIS

The reversible denaturant-induced unfolding of immunoglobulin molecule s has been analyzed by transverse urea gradient gel electrophoresis an d the effects that urea-induced unfolding exerts on the functional pro perties associated with their variable region, i.e. antigen binding an d idiotypic expression, have been determined by Western blot analysis. Results obtained from these experiments indicate that urea-induced un folding of the immunoglobulin molecule is a highly cooperative reversi ble process that occurs through a two-state transition with no accumul ation of intermediates. The unfolding transition has its midpoint at a bout 6.5 m urea and appears to be slow on the time scale of electropho resis. Folding intermediates in rapid equilibrium with the unfolded st ate 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 unfol ding of immunoglobulin molecules and demonstrate that the unfolding tr ansition leads to disappearance of both antigen binding and idiotypic expression, whereas the ability to interact with antibodies directed t o continuous epitopes of the variable region is preserved. After progr essive removal of the denaturing agent, the variable region refolds in to structures that regain the functional properties of the native conf ormation.