Department of Chemistry, University of Michigan, Ann Arbor, MI 48109, USA.
Anal Biochem. 2011 Apr 15;411(2):194-9. doi: 10.1016/j.ab.2011.01.009. Epub 2011 Jan 13.
A simple homogeneous assay for the detection of membrane permeabilization by antimicrobial peptides and synthetic copolymers is described. Liposomes encapsulating pyrroloquinoline quinone (PQQ), the prosthetic group of the apoenzyme glucose dehydrogenase (GDH), are used to detect membrane permeabilization by the antimicrobial peptides MSI-594 and MSI-78 as well as various synthetic antimicrobial copolymers in an optical microwell assay. PQQ-loaded liposomes and the peptide or copolymer are added to wells of a 96-well microtiter plate. If the integrity of the liposome is compromised, the PQQ encapsulated in the liposomes is released and available for activating the apoenzyme. The release of PQQ catalyzes a color change in the presence of apo-GDH, glucose, and the redox dye 1,6-dichlorophenol indophenol (DCPIP) that can be evaluated through a visual color change. For more quantitative measurements, the absorbance change over a 30min period was measured. The absorbance change is related to the activity and concentration for a given antimicrobial agent. Furthermore, by varying liposome compositions to include cholesterol, the potential toxicity of the peptide or polymer toward mammalian cells can be readily evaluated. The assay is simple and sensitive and will be useful for analyzing the membrane permeation/disruption properties of a host of antimicrobial peptides and synthetic polymers.
本文描述了一种用于检测抗菌肽和合成共聚物引起的膜通透性变化的简单均相测定法。采用包载吡咯喹啉醌（PQQ）的脂质体作为探针，检测抗菌肽 MSI-594 和 MSI-78 以及各种合成抗菌共聚物对细胞膜的通透性。将 PQQ 负载的脂质体与肽或共聚物加入 96 孔微量滴定板的孔中。如果脂质体的完整性受到破坏，包封在脂质体中的 PQQ 被释放出来，可用于激活脱辅基酶。在存在脱辅基葡萄糖脱氢酶（apo-GDH）、葡萄糖和氧化还原染料 1,6-二氯苯酚靛酚（DCPIP）的情况下，PQQ 的释放会催化颜色变化，可通过肉眼观察颜色变化进行评估。对于更定量的测量，可以在 30 分钟内测量吸光度变化。吸光度变化与给定抗菌剂的活性和浓度有关。此外，通过改变脂质体的组成以包含胆固醇，可以很容易地评估肽或聚合物对哺乳动物细胞的潜在毒性。该测定方法简单灵敏，将有助于分析一系列抗菌肽和合成聚合物的膜通透性/破坏特性。
