The thermal stability of immunoglobulin: Unfolding and aggregation of a multi-domain protein

The denaturation of immunoglobulin G was studied by different calorimetric methods and circular dichroism spectroscopy. The thermogram of the immunogl obulin showed two main transitions that are a superimposition of distinct d enaturation steps. It was shown that the two transitions have different sen sitivities 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 sensiti ve to heat treatment, whereas the F-c fragment is most sensitive to decreas ing pH. The transitions were independent, and the unfolding was immediately followed by an irreversible aggregation step. Below the unfolding temperat ure, 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 comple tely 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.