Precise detection of pH inside large unilamellar vesicles using membrane-impermeable dendritic porphyrin-based nanoprobes.
Precise detection of pH inside large unilamellar vesicles using membrane-impermeable dendritic porphyrin-based nanoprobes.
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DOI：
10.1016/j.ab.2009.02.023
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发表时间：
2009-05-15
影响因子：
2.9
通讯作者：
Arsköld SP
中科院分区：
文献类型：
--
作者：
Leiding T;Górecki K;Kjellman T;Vinogradov SA;Hägerhäll C;Arsköld SP
质子浓度梯度的准确实时测量对于膜结合酶质子易位的机制研究至关重要。在这里，我们报告了 pH 敏感荧光纳米探针 Glu3 的详细表征，它非常适合微室化生物系统中的 pH 测量。该探针是一种聚谷氨酸卟啉树枝状聚合物，其中多个羧酸盐末端确保其高水溶性并防止其扩散穿过磷脂膜。探针的 pK 处于生理 pH 范围内，其质子化可通过紫外-可见光谱区的吸光度或荧光进行比例跟踪。通过使用与电荷耦合器件 (CCD) 光谱仪耦合的半自动滴定系统，增强了探针的实用性，从而能够以毫秒时间分辨率实现快速、准确的滴定和系统的完整光谱覆盖。在存在生理学相关离子的情况下，在本体溶液以及大单层囊泡内部测量探针的 pK。人们发现 Glu3 是完全不可渗透膜的，其独特的光谱特征允许在封闭膜囊泡内进行 pH 测量，从而能够对跨膜蛋白进行定量机制研究。通过监测存在或不存在解偶联剂短杆菌肽的大囊泡中质子通过磷脂双层的泄漏率来证明探针的性能。总体而言，作为生物质子易位测量的探针，Glu3 优于市售 pH 指示剂。
Accurate real-time measurements of proton concentration gradients are pivotal to mechanistic studies of proton translocation by membrane-bound enzymes. Here we report a detailed characterization of the pH-sensitive fluorescent nanoprobe Glu3, which is well suited for pH measurements in microcompartmentalized biological systems. The probe is a polyglutamic porphyrin dendrimer in which multiple carboxylate termini ensure its high water solubility and prevent its diffusion across phospholipid membranes. The probe’s pK is in the physiological pH range, and its protonation can be followed ratiometrically by absorbance or fluorescence in the ultraviolet-visible spectral region. The usefulness of the probe was enhanced by using a semiautomatic titration system coupled to a charge-coupled device (CCD) spectrometer, enabling fast and accurate titrations and full spectral coverage of the system at millisecond time resolution. The probe’s pK was measured in bulk solutions as well as inside large unilamellar vesicles in the presence of physiologically relevant ions. Glu3 was found to be completely membrane impermeable, and its distinct spectroscopic features permit pH measurements inside closed membrane vesicles, enabling quantitative mechanistic studies of membrane-spanning proteins. Performance of the probe was demonstrated by monitoring the rate of proton leakage through the phospholipid bilayer in large vesicles with and without the uncoupler gramicidin present. Overall, as a probe for biological proton translocation measurements, Glu3 was found to be superior to the commercially available pH indicators.
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