Determination of acid dissociation constants of peptide side-chain functional groups by two-dimensional NMR

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.