Deuterium-Enhanced Raman Spectroscopy for Histidine pKa Determination in a pH-Responsive Hydrogel
Deuterium-Enhanced Raman Spectroscopy for Histidine pKa Determination in a pH-Responsive Hydrogel
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DOI：
10.1016/j.bpj.2020.09.011
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发表时间：
2020-11-03
影响因子：
3.4
通讯作者：
Akerfeldt, Karin S.
中科院分区：
文献类型：
--
作者：
Braun, Gabriel A.;Pogostin, Brett H.;Akerfeldt, Karin S.
我们在这里报告的方法测定pK（a）的组氨酸在复杂或非均相系统既不适合固态也不解决NMR光谱。小心合成芴基甲氧基羰基-和三苯甲基保护的C2-氘代组氨酸产生振动探针-装备的氨基酸，其可以容易地并入通过标准固相方法可获得的任何肽中。这种C2-D探针独特的拉曼活性伸缩振动的频率是组氨酸质子化状态的明确报告者。我们在这里调查的pH敏感的肽，自组装形成水凝胶在中性pH值。的pK（a）的肽中，这可能是负责这种pH值依赖性的行为，单独的组氨酸残基，不能通过NMR光谱研究，因为超分子，软性质的凝胶。然而，在合成含C2-氘代组氨酸的肽后，我们能够使用拉曼差光谱法在整个pH滴定过程中跟踪组氨酸的质子化状态，从而精确地确定感兴趣的pK（a）。
We report here a method for the determination of the pK(a) of histidine in complex or heterogeneous systems amenable to neither solid-state nor solution NMR spectroscopy. Careful synthesis of a fluorenylmethyloxycarbonyl- and tritylprotected, C2-deuterated histidine produces a vibrational-probe-equipped amino acid that can readily be incorporated into any peptide accessible by standard solid-phase methods. The frequency of the unique, Raman-active stretching vibration of this C2-D probe is a clear reporter of the protonation state of histidine. We investigate here a pH-sensitive peptide that self-assembles to form a hydrogel at neutral pH. The pK(a) of the lone histidine residue in the peptide, which is likely responsible for this pH-dependent behavior, cannot be investigated by NMR spectroscopy because of the supramolecular, soft nature of the gel. However, after synthesizing a C2-deuterated-histidine-containing peptide, we were able to follow the protonation state of histidine throughout a pH titration using Raman difference spectroscopy, thereby precisely determining the pK(a) of interest.