结合金 (III.) 对牛血清白蛋白不同构象的 Luminophore 形成
Summary
本文介绍了研究金阳离子 (III.) 对牛血清白蛋白的各种构象和构象依赖性独特的 bsa-Au 荧光的表征的协议。
Abstract
所提出的协议的目的是研究金 (iii) 对 bsa 的结合过程, 产生构象变化诱导红荧光 (λem = 640 nm) 的 bsa-Au (iii) 配合物。该方法调整 ph 值, 表明红荧光的出现与 BSA 构象的 ph 诱导的平衡过渡相关。红色荧光 BSA-Au (III.) 配合物只能形成与 pH 值在9.7 或以上的调整, 这对应于 “A 形” 构象的 BSA。介绍了将 BSA 调整为金摩尔比的协议, 并对金 (III.) 结合过程的时间过程进行了监测。每 BSA (III) 的最小数目, 以产生红色荧光, 小于七。我们描述了该协议的步骤, 以说明在 BSA 中存在多个 Au (III) 绑定站点。首先, 通过添加铜 (二) 或镍 (镍 (ii)) 阳离子其次为 au (iii), 这种方法揭示了一个结合点的非盟 (iii), 这不是红色荧光。其次, 通过硫醇封堵剂对 BSA 进行改性, 揭示了另一种 nonfluorophore 形成的金 (III.) 结合部位。第三, 通过对二硫化物键的劈裂和封盖改变 BSA 构象, 说明可能的 Au (III.) 结合点。该协议描述, 以控制 BSA 构象和 Au (III) 结合, 可以广泛应用于研究其他蛋白质和金属阳离子的相互作用。
Introduction
一种白蛋白-金化合物, 呈紫外 (紫外) 兴奋的红色荧光, 具有显著的斯托克斯移位, 最初是由谢等 al合成的。1. 独特和稳定的红色荧光可以在诸如传感2、3、4、成像5、6、7或纳米医学8等领域找到各种应用. ,9,10,11,12,13。近年来,14、15、16等纳米科学领域的研究人员对此化合物进行了广泛的研究。BSA-au 化合物被解释为 au25簇。所提出方法的目的是对该化合物进行详细的研究, 了解红荧光的起源。通过采用所提出的方法, 可以说明多个 au 结合点的存在, 以及荧光的起源, 替代非盟25簇的单点核。同样的方法也可以用来研究其他蛋白质17,18,19与 Au (III) 的复合物如何改变它们的内在荧光特性。
红荧光 bsa-au 化合物的合成需要对 bsa 的摩尔比 (bsa: au) 进行狭义的控制, 以最大限度地提高荧光强度和激发发射图 (EEM)20中峰值的位置。可以证明, 有多个绑定站点存在于 Au (III) 上, 包括天门冬片段 (或 Asp 片段, 在 BSA N 终点的前四个氨基酸残留物)21,22。34次氨基酸的 BSA (Cys-34) 也显示, 以协调 Au (iii) 和参与的机制, 红色荧光 ([Cys34 上限-BSA]-Au (iii))20。在裂解所有的胱氨酸-胱氨酸二硫化物键和上限所有硫醇, 红色荧光不产生 ([全硫醇上限-BSA]-Au (III))。这表明了胱氨酸-胱氨酸二硫化物键作为非盟 (III.) 结合部位产生红色荧光的必要性。
蛋白质化学技术还没有被广泛用于研究纳米科学群落中的 BSA (III.) 配合物。然而, 使用这些技术来了解这些复合体的某些方面, 以及对 BSA 中的非盟 (III) 结合点有详细的了解是很有价值的。本文旨在展示其中的一些技术。
Protocol
Representative Results
Discussion
在 pH 值为12的白蛋白 (III.) 化合物中制备的红色荧光在λem的发射波长 = 640 毫微米当激发与紫外 (紫外) 光λ前= 365 毫微米 (图 1A)。红色荧光的出现是一个缓慢的过程, 将需要几天在室温下增加到最大强度。在37摄氏度的反应将产生最佳的结果, 虽然更高的温度可以用来更快地产生红色荧光。蛋白质的不可逆转的退化可能发生在温度在45°c23</su…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
东南亚承认杜克捐赠特别倡议基金, 富国法戈基金, PhRMA 基金会的支持, 以及北卡罗来纳州大学夏洛特的启动资金。
Materials
| Bovine Serum Albumin (BSA), 96% | Sigma-Aldrich | A5611 | |
| gold (III) chloride trihydrate, 99.9% | Sigma-Aldrich | 520918 | |
| Copper (II) chloride dihydrate, 99.999% | Sigma-Aldrich | 459097 | |
| Nickel (II) chloride hexahydrate, 99.9% | Sigma-Aldrich | 654507 | |
| N-Ethylmaleimide (NEM), >99.0% | Sigma-Aldrich | 4259 | |
| Tris(2-carboxyethyl)phosphine hydrochloride (TCEP), >98.0% | Sigma-Aldrich | C4706 | |
| Sodium hydroxide, >98.0% | Sigma-Aldrich | S8045 | |
| Urea, 99.5% | Chem-Implex Int'l | 30142 | |
| Phospate buffered saline (PBS) | Corning | MT21040CV | |
| Ammonium bicarbonate, 99.5% | Sigma-Aldrich | 9830 |
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