Self-Assembled Monolayer of a Pepstatin Fragment as a Sensing Element for Aspartyl Proteases
作者:Hiromi Kitano、Yoshinobu Makino、Hideaki Kawasaki、Yusuke Sumi
DOI:10.1021/ac0488558
日期:2005.3.1
A novel disulfide, which carried two pepstatin fragments at both ends, was prepared by the coupling of 11,11‘-dithiobisundecanoic acid (DTUA) with a fragment (Val-Val-Sta) carrying a n-hexyl end (Pepsta(h)). The compound obtained (DTUA-Pepsta(h)) formed a self-assembled monolayer (SAM) on a gold electrode and vacuum-evaporated gold thin film as proven by cyclic voltammetry and reflection absorption infrared spectroscopy, respectively. When the SAM-modified gold electrode was incubated with a solution of aspartyl protease, pepsin, a decrease in both anodic and cathodic peak currents and an increase in potential difference were observed in the cyclic voltamogram of hydroquinone as a probe, whereas a coexistence of free pepstatin fragment inhibited these phenomena, indicating the specific binding of pepsin to the fragment at the exterior of the SAM. The binding rate of the enzyme to the SAM was largely dependent on the surface density of the fragment moiety in the SAM. Furthermore, when the SAM of DTUA-Pepsta(h) on a gold colloid array deposited on an amino group-modified glass plate was immersed in a pepsin solution, absorption of the glass plate at 550 nm corresponding to a localized surface plasmon resonance of the gold colloid abruptly increased and slightly red-shifted, and a further addition of pepstatin A gradually decreased the absorbance. From the increasing and decreasing profiles of absorbance, the association constant (Kassoc) for pepsin with the fragment on the SAM was determined. Similar phenomena were observed upon immersion of the fragment-modified SAM in a solution of HIV-1 protease, suggesting a usability of the pepstatin fragment SAM for the detection and removal of the enzyme from biological fluids.
通过将 11,11'-二硫代二癸酸(DTUA)与带有正己基端(Pepsta(h))的片段(Val-Val-Sta)偶联,制备出了一种新型二硫化物,其两端带有两个 Pepstatin 片段。获得的化合物(DTUA-Pepsta(h))在金电极和真空蒸发的金薄膜上形成了自组装单层(SAM),这分别通过循环伏安法和反射吸收红外光谱法得到了证实。当 SAM 修饰的金电极与天冬氨酰蛋白酶--胃蛋白酶溶液共孵育时,在以对苯二酚为探针的循环伏安图中观察到阳极和阴极峰电流均减小,电位差增大,而游离胃蛋白酶片段的共存则抑制了这些现象,这表明胃蛋白酶与 SAM 外部的片段有特异性结合。酶与 SAM 的结合率在很大程度上取决于 SAM 中片段分子的表面密度。此外,当将沉积在氨基修饰玻璃板上的金胶体阵列上的 DTUA-Pepsta(h)SAM 浸入胃蛋白酶溶液中时,玻璃板在 550 纳米波长处与金胶体局部表面等离子体共振相对应的吸收率突然升高并轻微红移,而进一步加入胃蛋白酶 A 会使吸收率逐渐降低。根据吸光度的递增和递减曲线,确定了胃蛋白酶与 SAM 上片段的结合常数 (Kassoc)。将片段修饰的 SAM 浸入 HIV-1 蛋白酶溶液中也观察到了类似的现象,这表明胃蛋白酶片段 SAM 可用于检测和去除生物液体中的酶。