Gomez-Orellana I, Variano B, Miura-Fraboni J, Milstein S, Paton D R
Emisphere Technologies, Inc., Hawthorne, New York 10532, USA.
Protein Sci. 1998 Jun;7(6):1352-8. doi: 10.1002/pro.5560070611.
The thermal denaturation of recombinant human growth hormone (rhGH) was studied by differential scanning calorimetry and circular dichroism spectroscopy (CD). The thermal unfolding is reversible only below pH 3.5, and under these conditions a single two-state transition was observed between 0 and 100 degrees C. The magnitudes of the deltaH and deltaCp of this transition indicate that it corresponds to a partial unfolding of rhGH. This is also supported by CD data, which show that significant secondary structure remains after the unfolding. Above pH 3.5 the thermal denaturation is irreversible due to the aggregation of rhGH upon unfolding. This aggregation is prevented in aqueous solutions of alcohols such as n-propanol, 2-propanol, or 1,2-propanediol (propylene glycol), which suggests that the self-association of rhGH is caused by hydrophobic interactions. In addition, it was found that the native state of rhGH is stable in relatively high concentrations of propylene glycol (up to 45% v/v at pH 7-8 or 30% at pH 3) and that under these conditions the thermal unfolding is cooperative and corresponds to a transition from the native state to a partially folded state, as observed at acidic pH in the absence of alcohols. In higher concentrations of propylene glycol, the tertiary structure of rhGH is disrupted and the cooperativity of the unfolding decreases. Moreover, the CD and DSC data indicate that a partially folded intermediate with essentially native secondary structure and disordered tertiary structure becomes significantly populated in 70-80% propylene glycol.
采用差示扫描量热法和圆二色光谱法（CD）研究了重组人生长激素（rhGH）的热变性。热解折叠仅在pH 3.5以下是可逆的，在这些条件下，在0至100摄氏度之间观察到单一的两态转变。该转变的ΔH和ΔCp大小表明它对应于rhGH的部分解折叠。CD数据也支持这一点，该数据表明解折叠后仍保留大量二级结构。在pH 3.5以上，由于rhGH解折叠时的聚集，热变性是不可逆的。在诸如正丙醇、异丙醇或1,2 - 丙二醇（丙二醇）等醇的水溶液中可防止这种聚集，这表明rhGH的自缔合是由疏水相互作用引起的。此外，发现rhGH的天然状态在相对高浓度的丙二醇中是稳定的（在pH 7 - 8时高达45% v/v，在pH 3时为30%），并且在这些条件下热解折叠是协同的，对应于从天然状态到部分折叠状态的转变，这与在没有醇存在的酸性pH下观察到的情况相同。在更高浓度的丙二醇中，rhGH的三级结构被破坏，解折叠的协同性降低。此外，CD和DSC数据表明，在70 - 80%的丙二醇中，具有基本天然二级结构和无序三级结构的部分折叠中间体大量出现。
