Abstract
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.
Highlights
“he reversible denaturant-induced unfolding ofim- in the interior hydrophobic region between the P-sheets
1988).Specific refolding procedures are necessary for the proagent, the variable region refolds into structures that duction of active recombinant whole Ig molecules expressed in regain the functional propertioefsthe native conforma-Escherichia coli and obtained in insoluble form as inclusion tion
To characterize theurea-induced unfoldingtransitions of in- The native molecule exhibits an unfolding pattern similar to tact Ig molecules and respective subunits, including F(ab'I2, that obtained with the whole G3 molecule, indicating a twostate folding transition
Summary
Native protein, unfolding occurs only a t a relatively high denaturant concentration and is observed in an abrupt transition with the followingmodifications.Tris acetate buffe(r0.05 M, pH 8.0)was that hasits midpoint at about 6.5 M urea Prior to this transiused to prepare 1.5-mm-thick polyacrylamide gels containing a hori- tion, there isno indication of any partialunfolding, as demonzontal linear gradient of 0-8 M urea and a compensatory inverse gradient of 167.5% acrylamide. Blotto (2.5% non-fat dry milk, 2.5% liquid gelatin, 0.05% Tween 20, 0.01%thimerosal, 0.001%antifoamA) for 4 ha t 4 "C.Nitrocellulose was washed with T-BBS and incubated overnight at 4 "C with BBS termediatesinrapid equilibrium withthe unfolded state These results show that, partially folded intermedicontaining 10% Blotto and one of the following reagents: (i)soluble CD4 ates cannot be detected during the unfolding of whole Ig moland rabbit polyclonal anti-CD4 antibodies, (i) a rabbit anti-idiotypic ecules, the refolding reaction occurs through different compact preparation, or (iii) rabbit anti-CDR peptide antibodies.
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