Almeida Paulo F, Pokorny Antje
Department of Chemistry and Biochemistry, University of North Carolina Wilmington, Wilmington, NC, USA.
Methods Mol Biol. 2010;618:155-69. doi: 10.1007/978-1-60761-594-1_11.
Measurement of binding and activity of antimicrobial and cytolytic amphipathic peptides on membranes is essential to understanding their function and cell specificity. The use of model systems has provided a wealth of information on the interactions of amphipathic peptides with membranes. Binding of peptides to membranes can be monitored by measuring Förster resonance energy transfer from a Trp residue on the peptide to a lipid fluorophore incorporated in the membrane. Especially for peptides that perturb or disrupt the membrane, it is advantageous to perform these measurements as a function of time, rather than in steady state. The activity of these amphipathic peptides toward model membranes is usually measured by dye efflux kinetics. One of those methods, based on self-quenching of carboxyfluorescein, is described here, together with a discussion of caveats and pitfalls of the corresponding analysis and interpretation.
测量抗菌和细胞溶解两亲性肽在膜上的结合和活性对于理解它们的功能和细胞特异性至关重要。模型系统的使用提供了大量关于两亲性肽与膜相互作用的信息。肽与膜的结合可以通过测量从肽上的色氨酸残基到掺入膜中的脂质荧光团的Förster共振能量转移来监测。特别是对于扰乱或破坏膜的肽，将这些测量作为时间的函数进行，而不是在稳态下进行，是有利的。这些两亲性肽对模型膜的活性通常通过染料流出动力学来测量。这里描述了其中一种基于羧基荧光素自猝灭的方法，同时讨论了相应分析和解释的注意事项和陷阱。
