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 CalphaH 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 (TOCSY) 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 CepsilonH2 protons adjacent to the four side-chain ammonium groups, which overlap in the one-dimensional spectrum, were resolved using the CalphaH-CepsilonH2 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 pK(A)s of the Lys(1), Lys(5), Lys(13), and Lys(14) 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: K-A(Lys(1))/K-A(Lys(5)) = 0.663 +/- 0.009, K-A(Lys(1))/K-A(Lys(13)) = 0.703 +/- 0.014, and K-A(Lys(1))/K-A(Lys(14)) = 0.910 +/- 0.009, which correspond to relative acidities for the side-chain ammonium groups of Lys(1), Lys(5), Lys(13), and Lys(14) 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-pa titration curves were obtained for the CbetaH2 protons adjacent to the two cysteine thiol groups using one-dimensional subspectra taken from TOCSY spectra measured as a function of pH, The pK(A)s of the Cys(3) and Cys(13) thiol groups were determined to be 9.21 +/- 0.07 and 8.60 +/- 0.06, respectively, The relative acid dissociation constants (K-A(Cys(3))/(K-A(Cys(13))) were found to be 0.21 +/- 0.06 by the pH-independent calculation procedure.