Vermeer A W, Norde W
Laboratory for Physical Chemistry, Wageningen Agricultural University, Dreijenplein 6, 6703 HB Wageningen, The Netherlands.
Biophys J. 2000 Jan;78(1):394-404. doi: 10.1016/S0006-3495(00)76602-1.
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.
通过不同的量热法和圆二色光谱法研究了免疫球蛋白G的变性。免疫球蛋白的热谱图显示出两个主要转变，这是不同变性步骤的叠加。结果表明，这两个转变对温度和pH值变化具有不同的敏感性。这两个峰分别代表IgG分子的F(ab)和F(c)片段。F(ab)片段对热处理最敏感，而F(c)片段对pH值降低最敏感。这些转变是相互独立的，并且变性之后紧接着是一个不可逆的聚集步骤。在变性温度以下，变性是整个变性过程中的速率决定步骤。在较高温度下，存在相对高浓度的（部分）变性IgG分子，聚集速率非常快，以至于IgG分子在完全变性之前就被锁定在聚集体中。此外，形成的聚集体的结构取决于变性方法。IgG的圆二色光谱也受到热处理和低pH处理的强烈影响。结果表明，量热法观察到的变性转变与圆二色性得出的二级结构变化之间存在很强的相关性。在热诱导和低pH诱导的变性之后，仍有相当一部分二级结构保留下来。
