Abstract
The toolset of mass spectrometry (MS) is still expanding, and the number of metal ion complexes researched this way is growing. The Cu(II) ion forms particularly strong peptide complexes of biological interest which are frequent objects of MS studies, but quantitative aspects of some reported results are at odds with those of experiments performed in solution. Cu(II) complexes are usually characterized by fast ligand exchange rates, despite their high affinity, and we speculated that such kinetic lability could be responsible for the observed discrepancies. In order to resolve this issue, we selected peptides belonging to the ATCUN family characterized with high and thoroughly determined Cu(II) binding constants and re-estimated them using two ESI-MS techniques: standard conditions in combination with serial dilution experiments and very mild conditions for competition experiments. The sample acidification, which accompanies the electrospray formation, was simulated with the pH–jump stopped-flow technique. Our results indicate that ESI-MS should not be used for quantitative studies of Cu(II)–peptide complexes because the electrospray formation process compromises the entropic contribution to the complex stability, yielding underestimations of complex stability constants.
Highlights
Complexation to peptides is proposed to play important roles inCu(II) physiology and toxicology
Two N-terminal sequence motifs, Xaa-His and Xaa-Zaa-His, provide the highest Cu(II) complex affinities by virtue of synergistic formation of chelate rings involving peptide nitrogen atoms (Figure 1).[18−20] The logarithmic conditional stability constants at physiological pH 7.4, log CK7.4, for Xaa-His complexes are in the range of 12.5−13, while those of Xaa-Zaa-His complexes range from 12.3 to ca. 15.21,22 The latter are known as ATCUN or NTS complexes.[18]
The dissociation of complexes was monitored by serial dilutions of both reagents
Summary
Complexation to peptides is proposed to play important roles inCu(II) physiology and toxicology. It elicits toxicity and probably gets detoxified by some variants of Aβ peptides[6,7] and protamine HP28−10 and likely participates in the antifungal action of histatins, salivary antimicrobial peptides.[11] A recent study indicated that more peptides with such properties remain to be identified in human proteome.[12] Peptides have been used extensively to model Cu(II) binding to its transport proteins, such as albumin and hCtr[1] membrane transporter,[13−15] and synaptic proteins, such as prions, APP, and αsynuclein.[16,17] Two N-terminal sequence motifs, Xaa-His and Xaa-Zaa-His (where Xaa is any α-amino acid except of Cys, and Zaa is any α-amino acid except of Cys or Pro), provide the highest Cu(II) complex affinities by virtue of synergistic formation of chelate rings involving peptide nitrogen atoms (Figure 1).[18−20] The logarithmic conditional stability constants at physiological pH 7.4, log CK7.4, for Xaa-His complexes are in the range of 12.5−13, while those of Xaa-Zaa-His complexes range from 12.3 to ca. Peptides have been used extensively to model Cu(II) binding to its transport proteins, such as albumin and hCtr[1] membrane transporter,[13−15] and synaptic proteins, such as prions, APP, and αsynuclein.[16,17] Two N-terminal sequence motifs, Xaa-His and Xaa-Zaa-His (where Xaa is any α-amino acid except of Cys, and Zaa is any α-amino acid except of Cys or Pro), provide the highest Cu(II) complex affinities by virtue of synergistic formation of chelate rings involving peptide nitrogen atoms (Figure 1).[18−20] The logarithmic conditional stability constants at physiological pH 7.4, log CK7.4, for Xaa-His complexes are in the range of 12.5−13, while those of Xaa-Zaa-His complexes range from 12.3 to ca. 15.21,22 The latter are known as ATCUN or NTS complexes.[18]
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