Multistep denaturation and hierarchy of disulfide bond cleavage of a monoclonal antibody.
A humanized, monoclonal antibody of the IgG4 class was treated with SDS at room temperature. Analysis using SDS-PAGE revealed the progressive formation of a series of denaturation intermediates, of increasing apparent molecular weights. The detectable species migrated with apparent molecular weights of 137, 152, 162, 182, 196, and 210 kDa. Antibody not incubated in SDS had an apparent molecular weight of 137 kDa, while boiling in SDS provoked the immediate conversion of all antibody to the slowest migrating form (210 kDa). A study designed to test if all rungs of the ladder were equally cleavable by mild treatment with mercaptoethanol revealed small amounts of a novel form, and conditions were devised to generate large amounts of this novel form. The novel form, before full denaturation, migrated at the position of unheated, intact antibody (137 kDa), while full denaturation in SDS converted the novel form to a species migrating at about 190 kDa. The novel form, before full denaturation, was identified as HHL ***L, where the asterisks indicate noncovalent binding of one light chain to HHL. This noncovalent complex remains intact during SDS-PAGE. The novel form, after full denaturation, was identified as HHL. This study, with earlier studies, reveals that different pathways of reductive cleavage are taken by different antibodies, and that the most sensitive disulfide bond is different for different antibodies. Our results on antibody denaturation serve as a warning to those who use two-dimensional gels for the analysis of antibodies.
Brody
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Athena Neurosciences, South San Francisco, California 94080, USA.
D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000302:isolation & purification; D001665:Binding Sites; D004220:Disulfides / Q000737:chemistry; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007074:Immunoglobulin G / Q000737:chemistry / Q000302:isolation & purification; D051379:Mice; D015394:Molecular Structure; D008970:Molecular Weight; D010084:Oxidation-Reduction; D011489:Protein Denaturation; D012967:Sodium Dodecyl Sulfate; D013997:Time Factors
D000911:Antibodies, Monoclonal; D004220:Disulfides; D007074:Immunoglobulin G; D012967:Sodium Dodecyl Sulfate