Høiberg-Nielsen Rasmus, Fuglsang Claus C, Arleth Lise, Westh Peter
Department of Life Sciences and Chemistry, Roskilde University, Building 18.1, P.O. Box 260, DK-4000 Roskilde, Denmark.
Biochemistry. 2006 Apr 18;45(15):5057-66. doi: 10.1021/bi0522955.
The kinetics of thermally induced aggregation of the glycoprotein Peniophora lycii phytase (Phy) and a deglycosylated form (dgPhy) was studied by dynamic (DLS) and static (SLS) light scattering. This provided a detailed insight into the time course of the formation of small aggregates ( approximately 10-100 molecules) of the enzyme. The thermodynamic stability of the two forms was also investigated using scanning calorimetry (DSC). It was found that the glycans strongly promoted kinetic stability (i.e., reduced the rate of irreversible denaturation) while leaving the equilibrium denaturation temperature, T(d), defined by DSC, largely unaltered. At pH 4.5-5.0, for example, dgPhy aggregated approximately 200 times faster than Phy, even though the difference in T(d) was only 1-3 degrees C. To elucidate the mechanism by which the glycans promote kinetic stability, we measured the effect of ionic strength and temperature on the aggregation rate. Also, the second virial coefficients (B(22)) for the two forms were measured by SLS. These results showed that the aggregation rate of Phy scaled with the concentration of thermally denatured protein. This suggested first-order kinetics with respect to the concentration of the thermally denatured state. A similar but less pronounced correlation was found for dgPhy, and it was suggested that while the aggregation process for the deglycosylated form is dominated by denatured protein, it also involves a smaller contribution from associating molecules in the native state. The measurements of B(22) revealed that dgPhy had slightly higher values than Phy. This suggests that dgPhy interacts more favorably with the buffer than Phy and hence rules out strong hydration of the glycans as the origin of their effect on the kinetic stability. On the basis of this and the effects of pH and ionic strength, we suggest that the inhibition of aggregation is more likely to depend on steric hindrance of the glycans in the aggregated form of the protein.
通过动态光散射（DLS）和静态光散射（SLS）研究了枸杞拟层孔菌糖蛋白植酸酶（Phy）和去糖基化形式（dgPhy）的热诱导聚集动力学。这为深入了解该酶小聚集体（约10 - 100个分子）形成的时间进程提供了详细信息。还使用扫描量热法（DSC）研究了这两种形式的热力学稳定性。结果发现，聚糖强烈促进了动力学稳定性（即降低了不可逆变性的速率），而由DSC定义的平衡变性温度T(d)基本未改变。例如，在pH 4.5 - 5.0时，尽管T(d)的差异仅为1 - 3℃，但dgPhy的聚集速度比Phy快约200倍。为了阐明聚糖促进动力学稳定性的机制，我们测量了离子强度和温度对聚集速率的影响。此外，通过SLS测量了这两种形式的第二维里系数（B(22)）。这些结果表明，Phy的聚集速率与热变性蛋白的浓度成比例。这表明对于热变性状态的浓度而言是一级动力学。对于dgPhy发现了类似但不太明显的相关性，并且有人提出，虽然去糖基化形式的聚集过程以变性蛋白为主导，但它也涉及天然状态下缔合分子的较小贡献。对B(22)的测量表明，dgPhy的值略高于Phy。这表明dgPhy与缓冲液的相互作用比Phy更有利，因此排除了聚糖的强水合作用是其对动力学稳定性产生影响的根源。基于此以及pH和离子强度的影响，我们认为聚集的抑制更可能取决于聚糖在蛋白质聚集形式中的空间位阻。
