Department of Applied Biological Science, Tokyo University of Science, Noda, Chiba 278-8510, Japan.
Anal Biochem. 2011 Dec 15;419(2):173-9. doi: 10.1016/j.ab.2011.08.019. Epub 2011 Aug 18.
A foam fractionation apparatus was prepared to aid protein separation at the gas-liquid interface. Using lysozyme as a model protein, we investigated the alteration of enzymatic and optical activities through foaming. The lysozyme transferred to the gaseous nitrogen phase after 5 min of bubbling with no exogenous detergent. The bacteriolytic and optical activities of lysozyme from the foamate were nearly equivalent to those of the original lysozyme. This result indicated that lysozyme did not irreversibly denature during foam fractionation. We then performed protein separation using binary mixtures of lysozyme and α-amylase. When the two proteins were dissolved in bulk solution of pH 10.5, which is close to the isoelectric point (pI) of lysozyme (10.7), selective fractionation of lysozyme from the foam was observed. Indeed, this fractionation was identical to that from a single component solution of lysozyme. Similarly, selective fractionation of α-amylase was achieved in pH 3.0 buffer. Furthermore, circular dichroism (CD) and subsequent model fitting revealed that the protein had a reduced or nearly complete absence of α-helical content, whereas the amount of β-sheet structure and random coil was elevated in the buffer conditions that promoted protein adsorption. These results indicate that a pH-induced conformational transition might correlate with protein foaming.
我们制备了一种泡沫分馏装置，以帮助在气液界面进行蛋白质分离。我们以溶菌酶为模型蛋白，通过鼓泡研究了泡沫对酶活性和光学活性的影响。溶菌酶在氮气中鼓泡 5 分钟后转移到气态氮相中，没有使用外源性去污剂。泡沫层中溶菌酶的溶菌酶活性和光学活性与原始溶菌酶几乎相当。这表明溶菌酶在泡沫分馏过程中没有发生不可逆变性。然后，我们使用溶菌酶和α-淀粉酶的二元混合物进行蛋白质分离。当两种蛋白质溶解在接近溶菌酶等电点（pI）10.7 的 pH 10.5 的 bulk 溶液中时，观察到溶菌酶从泡沫中选择性地分离出来。事实上，这种分离与从单一成分溶菌酶溶液中的分离相同。同样，在 pH 3.0 缓冲液中也实现了α-淀粉酶的选择性分离。此外，圆二色性（CD）和随后的模型拟合表明，在促进蛋白质吸附的缓冲条件下，蛋白质的α-螺旋含量减少或几乎完全不存在，而β-折叠结构和无规卷曲的含量增加。这些结果表明，pH 诱导的构象转变可能与蛋白质起泡有关。
