MOLECULAR AGGREGATION AND INDUCED NUCLEATION IN PROTEIN CRYSTAL GROWTH

本课题主要利用动态光散射技术和交叉尼克尔斯法分析蛋白质晶体生长过程中诱导成核的动力学机制。本研究涉及的蛋白质分子有溶菌酶和高卡淀粉酶。现将crusad - nicols法对溶菌酶结晶的结果总结如下：(1)通过测量不同过饱和值下晶体出现的持续时间获得诱导时间；(2)通过计算诱导时间来评估界面能（gamma），其反比值与成核速率和过饱和速率成正比；(3)坐式和吊滴法结晶的gamma值（erg/cm^2）均为0.3。(4)微种子效应与种子晶体大小和过饱和度有关。塔卡淀粉酶结晶的结果总结如下：(1)在过饱和溶液中，经过一定的诱导次数后，扩散常数和平均粒径突然增加；(2)这种变化是随着过饱和值的增加而发生的，随后出现肉眼可见的晶体。因此，通过动态光散射技术的检测，我们可以得出结论，taka-淀粉酶的分子聚簇是在成核之前形成的。这两种蛋白质的结果表明，在分子量与塔卡淀粉酶一样大的情况下，预成核事件是可检测的。然而，由于缺乏溶解度的基本数据，更精确的实验是不可能的。因此，现在用双光束干涉法测量塔卡淀粉酶的溶解度。

This research project aimed at analyzing kinetic mechanisms in induced nucleation during the processes of protein crystal growth, mainly using dynamic light scattering techniques and crossed Nicols method. The protein molecules dealt with in the present work were lysozyme and taka-amylase.The results obtained for lysozyme crystallization using the crusaded-Nicols method are summarized in the following ; (1) the induction times were obtained by measuring the duration for the occurrence of crystals in varying supersaturating values, (2) interface energies (gamma) were evaluated from the calculation of the induction times, whose inverse values are proportional to the rates of nucleation, and supersaturation, (3) the gamma values (erg/cm^2) were 0.3 both for sitting and hanging drop methods for crystallization, (4) micro-seeding effects were found to be dependent on the sizes of seed crystals and supersaturations.The results obtained for taka-amylase crystallization are summarized in the following ; (1) diffusion constants and average particle sizes increased quite abruptly after certain induction times in supersaturated solution, (2) the occurrence of this cahnges was prompted with increasing supersaturation values, and followed by the appearance of crystals detectable by naked eyes. Therefore, one may concluded that the molecular clustering of taka-amylase was formed in prior to nucleation, as detected by the dynamic light scattering technique.These results available for the two types of proteins have shown that the pre-nucleation events are detectable, in case that the molecular weights are as large as in taka-amylase. However, more precise experiments are not possible, because the fundamental data of solubility are lacking. Thus, the solubility of taka-amylase is now measured by using a two-beam interferometric method.