The Nrf2 (69-84) peptide, with the amino acid sequence AFFAQLQLDEETGEFL, corresponds to residues 69 to 84 of the human Nrf2 protein. This 16-mer peptide contains the critical ETGE motif, which is a high-affinity binding site for the Kelch domain of the Keap1 protein. Structural studies, including X-ray crystallography, reveal that this peptide adopts a conformation featuring two anti-parallel strands connected by overlapping type I β-turns when bound to Keap1. The glutamate residues within the ETGE motif, specifically E79 and E82, form multiple specific interactions with residues in the Kelch domain's binding pocket, such as R415 and R483, while the peptide backbone makes additional contacts involving E78, T80, and F83.

Biophysical analyses, including surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC), have characterized the interaction. The equilibrium dissociation constant (K_D) for the binding of this peptide to the Keap1 Kelch domain in solution is approximately 20-24 nM. Deletion studies indicate that the minimal Nrf2 peptide sequence required for maintaining high-affinity Keap1 binding is a 9-mer sequence, LDEETGEFL (residues 76-84). This shorter peptide retains a β-hairpin conformation stabilized by intramolecular hydrogen bonds and exhibits moderate binding affinity (K_D ~352 nM), which can be significantly enhanced by N-terminal acetylation to remove a positive charge.

• Chen Y, Inoyama D, Kong AN, Beamer LJ, Hu L. Kinetic analyses of Keap1-Nrf2 interaction and determination of the minimal Nrf2 peptide sequence required for Keap1 binding using surface plasmon resonance. Chem Biol Drug Des. 2011 Dec;78(6):1014-21. doi: 10.1111/j.1747-0285.2011.01240.x. Epub 2011 Oct 31. PMID: 21920027; PMCID: PMC3218206.

三氟乙酸（Trifluoroacetic acid，简称 TFA）是高效液相色谱（简称 HPLC）纯化过程中常用的反离子。TFA 的存在可能会对肽的净重量、外观及溶解度产生影响，具体如下：

对净重量的影响：TFA 盐会增加产物的总质量。在大多数情况下，肽的含量占总重量的 80% 以上，剩余部分则为 TFA。

对溶解度的影响：TFA 盐通常能提高肽在水溶液中的溶解度。

在生物检测中的影响：对于大多数标准的体外检测，残留的 TFA 水平一般不会造成干扰。但需注意，在高灵敏度的细胞研究或生化研究中，需关注其存在可能带来的影响。

干品多肽的重量中不仅仅包含多肽，还包含有一些非肽的组份，如水、被吸收的溶剂、配位离子和盐等。肽的净含量是指肽在其中的重量百分比，这个百分比的数值范围很大，可能从50%到90%，取决于纯度、序列以及合成和纯化的方法，不要将肽的净含量和肽的纯度混为一谈，他们是两个完全不同的概念。纯度通常是通过HPLC测定的。纯度定义的是多肽样品中含正确序列的组分的百分比，而肽的净含量是指样品中肽类物质相对于总物质所占的百分比，肽的净含量通常是氨基酸组分分析或紫外分光法测定的，这个信息主要是在一些对肽的浓度很敏感的实验中，对计算肽的浓度是很重要的。