Fears Kenan P, Sivaraman Balakrishnan, Powell Gary L, Wu Yonnie, Latour Robert A
Department of Bioengineering, Clemson University Genomics Institute, Clemson University, Clemson, SC 29634, USA.
Langmuir. 2009 Aug 18;25(16):9319-27. doi: 10.1021/la901885d.
The bioactivity of enzymes that are adsorbed on surfaces can be substantially influenced by the orientation of the enzyme on the surface and adsorption-induced changes in the enzyme's structure. Circular dichroism (CD) is a powerful method for observing the secondary structure of proteins; however, it provides little information regarding the tertiary structure of a protein or its adsorbed orientation. In this study, we developed methods using side-chain-specific chemical modification of solvent-exposed tryptophan residues to complement CD spectroscopy and bioactivity assays to provide greater detail regarding whether changes in enzyme bioactivity following adsorption are due to adsorbed orientation and/or adsorption-induced changes in the overall structure. These methods were then applied to investigate how adsorption influences the bioactivity of hen egg white lysozyme (HEWL) and glucose oxidase (GOx) on alkanethiol self-assembled monolayers over a range of surface chemistries. The results from these studies indicate that surface chemistry significantly influences the bioactive state of each of these enzymes but in distinctly different ways. Changes in the bioactive state of HEWL are largely governed by its adsorbed orientation, while the bioactive state of adsorbed GOx is influenced by a combination of both adsorbed orientation and adsorption-induced changes in conformation.
吸附在表面的酶的生物活性会受到酶在表面的取向以及吸附诱导的酶结构变化的显著影响。圆二色性（CD）是观察蛋白质二级结构的有力方法；然而，它几乎无法提供有关蛋白质三级结构或其吸附取向的信息。在本研究中，我们开发了利用对溶剂暴露的色氨酸残基进行侧链特异性化学修饰的方法，以补充CD光谱和生物活性测定，从而更详细地了解吸附后酶生物活性的变化是由于吸附取向和/或吸附诱导的整体结构变化所致。然后将这些方法应用于研究在一系列表面化学条件下，吸附如何影响蛋清溶菌酶（HEWL）和葡萄糖氧化酶（GOx）在烷硫醇自组装单分子层上的生物活性。这些研究结果表明，表面化学显著影响这两种酶各自的生物活性状态，但方式明显不同。HEWL生物活性状态的变化很大程度上由其吸附取向决定，而吸附的GOx的生物活性状态则受吸附取向和吸附诱导的构象变化共同影响。
