title: The thermal stability of immunoglobulin: unfolding and aggregation of a multi-domain protein.
abstract: The denaturation of immunoglobulin G was studied by different calorimetric methods and circular dichroism spectroscopy. The thermogram of the immunoglobulin showed two main transitions that are a superimposition of distinct denaturation steps. It was shown that the two transitions have different sensitivities to changes in temperature and pH. The two peaks represent the F(ab) and F(c) fragments of the IgG molecule. The F(ab) fragment is most sensitive to heat treatment, whereas the F(c) fragment is most sensitive to decreasing pH. The transitions were independent, and the unfolding was immediately followed by an irreversible aggregation step. Below the unfolding temperature, the unfolding is the rate-determining step in the overall denaturation process. At higher temperatures where a relatively high concentration of (partially) unfolded IgG molecules is present, the rate of aggregation is so fast that IgG molecules become locked in aggregates before they are completely denatured. Furthermore, the structure of the aggregates formed depends on the denaturation method. The circular dichroism spectrum of the IgG is also strongly affected by both heat treatment and low pH treatment. It was shown that a strong correlation exists between the denaturation transitions as observed by calorimetry and the changes in secondary structure derived from circular dichroism. After both heat- and low-pH-induced denaturation, a significant fraction of the secondary structure remains.
authors.affiliation: Laboratory for Physical Chemistry, Wageningen Agricultural University, Dreijenplein 6, 6703 HB Wageningen, The Netherlands. Ronald.Vermeer.RV@BAYER-AG.DE
authors.forename: A W
authors.initials: AW
authors.lastname: Vermeer
authors.forename: W
authors.initials: W
authors.lastname: Norde
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry*; D002151:Calorimetry / Q000379:methods; D002942:Circular Dichroism; D006442:Glycated Hemoglobin / Q000276:immunology; D006358:Hot Temperature; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007074:Immunoglobulin G / Q000737:chemistry*; D007132:Immunoglobulin Isotypes / Q000737:chemistry; D051379:Mice; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D051381:Rats; D013816:Thermodynamics
chemical_list: D000911:Antibodies, Monoclonal; D006442:Glycated Hemoglobin; D007074:Immunoglobulin G; D007132:Immunoglobulin Isotypes