Rabenstein D L, Hari S P, Kaerner A
Department of Chemistry, University of California, Riverside 92521, USA.
Anal Chem. 1997 Nov 1;69(21):4310-6. doi: 10.1021/ac970675t.
An NMR method is described for determining residue-specific acid dissociation constants for peptides which contain more than one residue of the same acidic or basic amino acid. The method is based on using the differences in C alpha H proton chemical shifts which result from peptide sequence nearest-neighbor and possibly secondary structure effects to resolve resonances for carbon-bonded reporter protons adjacent to each side-chain acidic group in two-dimensional total correlation spectroscopy (TOC-SY) spectra. Acid dissociation constants were determined for each of the four lysine side-chain ammonium groups of the peptide Lys-Asn-Asn-Gln-Lys-Ser-Glu-Pro-Leu-Ile-Gly-Arg-Lys-Lys-Thr-NH2. Resonances for the C epsilon H2 protons adjacent to the four side-chain ammonium groups, which overlap in the one-dimensional spectrum, were resolved using the C alpha H-C epsilon H2 cross peaks in the TOCSY spectrum. Chemical shift-pH titration data were obtained for each lysine side-chain ammonium group from one-dimensional subspectra taken from two-dimensional TOCSY spectra measured as a function of pH. The pKAs of the Lys1, Lys5, Lys13, and Lys14 side-chain ammonium groups were determined to be 11.14 +/- 0.01, 10.95 +/- 0.01, 10.96 +/- 0.02, and 11.09 +/- 0.02, respectively. The chemical shift-pH titration data were also analyzed by a pH-independent procedure to obtain relative acid dissociation constants: KA(Lys1)/KA(Lys5) = 0.663 +/- 0.009, KA(Lys1)/KA(Lys13) = 0.703 +/- 0.014, and KA(Lys1)/KA(Lys14) = 0.910 +/- 0.009, which correspond to relative acidities for the side-chain ammonium groups of Lys1, Lys5, Lys13, and Lys14 of 1:1.508:1.422:1.099. To further demonstrate the utility of this method, acid dissociation constants were determined for the six acidic groups of the peptide Glu-Ala-Cys-Asn-Pro-Ala-Ala-Gly-Arg-His-Tyr-Ser-Cys-NH2. Chemical shift-pH titration curves were obtained for the C beta H2 protons adjacent to the two cysteine thiol groups using one-dimensional subspectra taken from TOCSY spectra measured as a function of pH. The pKAs of the CyS3 and Cys13 thiol groups were determined to be 9.21 +/- 0.07 and 8.60 +/- 0.06, respectively. The relative acid dissociation constants (KA(Cys3)/ (KA(Cys13)) were found to be 0.21 +/- 0.06 by the pH-independent calculation procedure.
本文描述了一种核磁共振（NMR）方法，用于测定含有一个以上相同酸性或碱性氨基酸残基的肽的残基特异性酸解离常数。该方法基于利用由于肽序列最近邻以及可能的二级结构效应导致的CαH质子化学位移差异，来解析二维全相关谱（TOCSY）谱中与每个侧链酸性基团相邻的碳键连接的报告质子的共振。测定了肽Lys-Asn-Asn-Gln-Lys-Ser-Glu-Pro-Leu-Ile-Gly-Arg-Lys-Lys-Thr-NH2的四个赖氨酸侧链铵基团中每个基团的酸解离常数。在一维谱中重叠的与四个侧链铵基团相邻的CεH2质子的共振，通过TOCSY谱中的CαH-CεH2交叉峰得以解析。从作为pH函数测量的二维TOCSY谱获取的一维子谱中，得到了每个赖氨酸侧链铵基团的化学位移-pH滴定数据。Lys1、Lys5、Lys13和Lys14侧链铵基团的pKa值分别测定为11.14±0.01、10.95±0.01、10.96±0.02和11.09±0.02。化学位移-pH滴定数据还通过一种与pH无关的程序进行分析，以获得相对酸解离常数：KA(Lys1)/KA(Lys5)=0.663±0.009，KA(Lys1)/KA(Lys13)=0.703±0.014，以及KA(Lys1)/KA(Lys14)=0.910±0.009，这对应于Lys1、Lys5、Lys13和Lys14侧链铵基团的相对酸度为1:1.508:1.422:1.099。为了进一步证明该方法的实用性，测定了肽Glu-Ala-Cys-Asn-Pro-Ala-Ala-Gly-Arg-His-Tyr-Ser-Cys-NH2的六个酸性基团的酸解离常数。使用从作为pH函数测量的TOCSY谱获取的一维子谱，得到了与两个半胱氨酸硫醇基团相邻的CβH2质子的化学位移-pH滴定曲线。CyS3和Cys13硫醇基团的pKa值分别测定为9.21±0.07和8.60±0.06。通过与pH无关的计算程序发现相对酸解离常数（KA(Cys3)/KA(Cys13)）为0.21±0.06。
