Chan H K, Au-Yeung K L, Gonda I
Department of Pharmaceutical Research and Development, Genentech, Inc. South San Francisco, California 94080, USA.
Pharm Res. 1996 May;13(5):756-61. doi: 10.1023/a:1016007818575.
To study the thermal stability of recombinant human deoxyribonuclease I (rhDNase) in aqueous solutions.
Differential scanning calorimetry (DSC) was used to measure the denaturation or melting temperature (T(m)) and enthalpy (H(m)) of rhDNase. The effects of denaturants (guanidine HCl and urea) and additives (mainly divalent cations and disaccharides) were investigated at pH 6-7.
The T(m) and H(m) of rhDNase in pure water were measured as 67.4 degrees C and 18.0 J/g respectively, values typical of globular proteins. The melting peak disappeared on re-running the sample after cooling to room temperature, indicating that the thermal denaturation was irreversible. The latter was due to the occurrence of aggregation accompanying the unfolding process of rhDNase. Size exclusion chromatography indicated that during heat denaturation, rhDNase formed soluble high molecular weight aggregates with a molecular size >300kD estimated by the void volume. Of particular interest are the divalent cations: Ca(2+) stabilizes rhDNase against thermal denaturation and elevates T(m) and H(m) while Mg(2+), Mn(2+) and Zn(2+) destabilize it. Sugars also stabilize rhDNase. As expected, denaturants destabilize the protein and lower the T(m) and H(m). All destabilization of rhDNase can be prevented by adding Ca(2+) to the solutions.
CaCl(2) and sugars were found to stabilize rhDNase against thermal denaturation while divalent cations, urea and guanidine HCl destabilize the protein. The effects could be explained by a mixture of mechanisms. For Ca(2+) the protective effect is believed to be due to an ordering of the rhDNase structure in its native state, and by prevention of breaking of a disulfide bridge, thus making it less susceptible to unfold under thermal stress.
研究重组人脱氧核糖核酸酶I（rhDNase）在水溶液中的热稳定性。
采用差示扫描量热法（DSC）测定rhDNase的变性或解链温度（T(m)）和焓（H(m)）。在pH 6 - 7条件下研究变性剂（盐酸胍和尿素）和添加剂（主要是二价阳离子和二糖）的影响。
rhDNase在纯水中的T(m)和H(m)分别测定为67.4℃和18.0 J/g，这是球状蛋白的典型值。冷却至室温后重新运行样品时，解链峰消失，表明热变性是不可逆的。后者是由于rhDNase展开过程中伴随发生聚集。尺寸排阻色谱表明，在热变性过程中，rhDNase形成了可溶性高分子量聚集体，根据空体积估计其分子大小>300kD。特别值得关注的是二价阳离子：Ca(2+)可稳定rhDNase使其抗热变性，并提高T(m)和H(m)，而Mg(2+)、Mn(2+)和Zn(2+)则使其不稳定。糖类也能稳定rhDNase。正如预期的那样，变性剂会使蛋白质不稳定并降低T(m)和H(m)。向溶液中添加Ca(2+)可防止rhDNase的所有不稳定情况。
发现CaCl(2)和糖类可稳定rhDNase使其抗热变性，而二价阳离子、尿素和盐酸胍会使蛋白质不稳定。这些影响可以用多种机制的混合来解释。对于Ca(2+)，保护作用被认为是由于rhDNase天然状态下结构的有序化，以及防止二硫键断裂，从而使其在热应激下更不易展开。
