Short peptides are not reliable models of thermodynamic and kinetic properties of the N‐terminal metal binding site in serum albumin

Sokołowska, Magdalena; Krężel, Artur; Dyba, Marcin; Szewczuk, Zbigniew; Bal, Wojciech

doi:10.1046/j.1432-1033.2002.02772.x

Cited by 97 publications

(137 citation statements)

References 42 publications

(70 reference statements)

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“…No abs ∆H for DAHK could be found, but for it has been determined for the analogue peptide KGHK [20], and based on the absolute values a cond ∆H of -0.5 kcal was calculated (see Materials and Methods). These values are relatively similar to the one determined here, and the larger difference in the case of DAHK versus KGHK can be explained by the different pKa values of the ligands [20,34]. (Table 6)..…”

Section: Resultssupporting

confidence: 85%

“…Moreover, human serum albumin (HSA) containing a DAHK motive has a lower affinity than DAHK, [32,34] and hence GHK is perhaps a stronger Cu 2+ chelator than HSA [40]. Moreover, the present measurements confirm clearly that either DAHK or GHK are much stronger Cu 2+ chelators by at least an order of magnitude than the peptide Aβ at pH 7.4.…”

Section: Resultssupporting

confidence: 59%

“…For GHK the pKa values of amine and histidine are equal to 7.85 and 6.45 [19], while for DAHK these values are 7.53 and 6.28 [34]. This seems not to fit with the experimental data.…”

Section: Resultsmentioning

confidence: 72%

“…The reported conditional dissociation constant of GHK and analogues of DAHK are about 1 x 10 -14 M [19,20,[31][32][33][34][35][36]. Although in a 100mM HEPES buffer the affinity will decrease to a app Kd of about 10 -12 M at pH 7.4 [26], this affinity is still too high to be measured directly by ITC.…”

Section: Resultsmentioning

confidence: 97%

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Thermodynamic study of Cu2+ binding to the DAHK and GHK peptides by isothermal titration calorimetry (ITC) with the weaker competitor glycine

et al. 2011

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The peptides Asp-Ala-His-Lys (DAHK) and Gly-His-Lys (GHK) are naturally occurring copper(II)-chelating motives in human serum and cerebrospinal fluid. Here the sensitive thermodynamic technique isothermal titration calorimetry (ITC) has been used to study the energetics of copper(II) binding to DAHK and GHK peptides in the presence of the weaker ligand glycine as a competitor. DAHK and GHK bind Cu(II) predominantly in a 1:1 stoichiometry with conditional dissociation constants (i.e. at pH 7.4, in the absence of the competing chelators glycine and HEPES buffer) of 2.6 +/-0.4 x 10 -14 M and of 7.0 +/-1.0 x 10 -14 M, respectively. Furthermore, the apparent ΔH values were measured and the number of protons released upon Cu(II) binding was determined by performing experiments in different buffers. This allowed us to determine the conditional ΔG, ΔH, and ΔS, i.e. corrected for the contributions of the weaker ligand glycine and the buffer at pH 7.4. We found that the entropic and enthalpic contributions to the Cu(II)-binding to GHK and DAHK are distinct, with a higher enthalpic contribution for GHK. The obtained thermodynamic parameters correspond well to those in the literature obtained by other techniques, suggesting that the use of the weaker ligand glycine as a competitor in ITC provides accurate data for Cu(II)-binding to high affinity peptides, which cannot be accurately determined without the use of a competitor ligand.3

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Section: Resultssupporting

confidence: 85%

Section: Resultssupporting

confidence: 59%

“…For GHK the pKa values of amine and histidine are equal to 7.85 and 6.45 [19], while for DAHK these values are 7.53 and 6.28 [34]. This seems not to fit with the experimental data.…”

Section: Resultsmentioning

confidence: 72%

Section: Resultsmentioning

confidence: 97%

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Thermodynamic study of Cu2+ binding to the DAHK and GHK peptides by isothermal titration calorimetry (ITC) with the weaker competitor glycine

et al. 2011

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“…Although the natural genetic code does not include aldehyde or ketone-containing amino acids, proteins generally exhibit some level of aldehyde or ketone content even in the absence of purposeful oxidation 9,10,24,25 . One of the many advantages of the O-ECAT AOD system is the availability of the 2D12.5 monoclonal antibody specific for a range of M-DOTA chelates 18 .…”

Section: Aod Labelingmentioning

confidence: 99%

Method to Site-Specifically Identify and Quantitate Carbonyl End Products of Protein Oxidation Using Oxidation-Dependent Element Coded Affinity Tags (O-ECAT) and NanoLiquid Chromatography Fourier Transform Mass Spectrometry

et al. 2006

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Protein oxidation is linked to cellular stress, aging, and disease. Protein oxidations that result in reactive species are of particular interest, since these reactive oxidation products may react with other proteins or biomolecules in an unmediated and irreversible fashion, providing a potential marker for a variety of disease mechanisms. We have developed a novel system to identify and quantitate, relative to other states, the sites of oxidation on a given protein. A specially designed Oxidation-dependent carbonyl-specific Element-Coded Affinity Mass Tag (O-ECAT), AOD, ((S)-2-(4-(2-aminooxy)-acetamido)-benzyl)-1, 4, 7, 10-tetraazacyclododecane-N, N′, N′′, N′′′-tetraacetic acid, is used to covalently tag the residues of a protein oxidized to aldehyde or keto end products.After proteolysis, the resulting AOD-tagged peptides are affinity purified, and analyzed by nanoLC-FTICR-MS, which provides high specificity in extracting co-eluting AOD mass pairs with a unique mass difference and affords relative quantitation based on isotopic ratios. Using this methodology, we have mapped the surface oxidation sites on a model protein, recombinant human serum albumin (rHSA) in its native form (as purchased) and after FeEDTA oxidation. A variety of modified amino acid residues including lysine, arginine, proline, histidine, threonine, aspartic and glutamic acids, were 3 found to be oxidized to aldehyde and keto end products. The sensitivity of this methodology is shown by the number of peptides identified, twenty peptides on the native protein and twenty-nine after surface oxidation using FeEDTA and ascorbate. All identified peptides map to the surface of the HSA crystal structure validating this method for identifying oxidized amino acids on protein surfaces. In relative quantitation experiments between FeEDTA oxidation and native protein oxidation, identified sites showed different relative propensities towards oxidation independent of amino acid residue. We expect to extend this methodology to study disease-related oxidation systems.

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A Functional Role for Aβ in Metal Homeostasis? N‐Truncation and High‐Affinity Copper Binding

et al. 2015

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Accumulation of the b-amyloid (Ab)p eptide in extracellular senile plaques rich in copper and zinc is adefining pathological feature of Alzheimer'sd isease (AD). The Ab1-x( x= 16/28/40/42) peptides have been the primary focus of Cu II binding studies for more than 15 years;h owever,t he Ntruncated Ab4-42 peptide is am ajor Ab isoform detected in both healthy and diseased brains,a nd it contains an ovel Nterminal FRH sequence.Proteins with His at the thirdposition are knowntobind Cu II avidly,with conditional log Kvalues at pH 7.4 in the range of 11.0-14.6, whichi sm uch higher than that determined for Ab1-x peptides.B yu sing Ab4-16 as am odel, it was demonstrated that its FRH sequence stoichiometrically binds Cu II with aconditional K d value of 310 À14 m at pH 7.4, and that both Ab4-16 and Ab4-42 possess negligible redox activity.Combined with the predominance of Ab4-42 in the brain, our results suggest ap hysiological role for this isoform in metal homeostasis within the central nervous system.Alzheimer' sd isease (AD) is aneurodegenerative condition characterized by progressive cognitive decline and cerebral deposition of fibrillar plaques comprised of the b-amyloid (Ab)peptide.[1] There is compelling evidence that in AD,the brain undergoes widespread oxidative stress,w hich has been linked with unusually high concentrations of redox-active transition metals,p articularly Cu, within amyloid plaques. [2] Thefirst protein sequencing studies of the plaque core of AD patients identified asignificant proportion of Ab peptides with "ragged" Ntermini, with Ab4-x isoforms accounting for more than 60 %o fb rain amyloid. [3,4] More recent mass spectrometry analyses confirmed these seminal findings, demonstrating that Ab4-42 and Ab1-42 are the dominant isoforms present in the hippocampus and cortex of sporadic AD patients,a sw ell as in healthy controls. [5,6] Although we have previously noted that Ab4-x peptides contain the amino-terminal copper and nickel (ATCUN,H 2 N-XaaYaa-His-) motif, [7] which enables high affinity Cu II binding via a{ NH 2 Xaa ,N ÀYaa ,N ÀHis ,N Im His }c oordination sphere, [8] interest has remained focused on elucidating the Cu II coordination and affinity of Ab1-x peptides. [9,10] In this study,w ef ully characterize the Cu II binding properties of Ab4-16 by using ar ange of spectroscopic and potentiometric techniques.W ed emonstrate that the fused (5,5,6)-membered chelate rings formed by the first three residues of Ab4-x provide aC u II binding site with ac onditional log K value more than three orders of magnitude higher than that reported for Ab1-x peptides and thirty times higher than that of human serum albumin (HSA), which also shares the ATCUN motif.[11] TheA b4-16 peptide contains asecond Cu II binding site,fully separated from the N-terminal site,b ut with an affinity that is lower by seven orders of magnitude.We began by characterizing the pH dependence of Cu II coordination by Ab4-16 by UV/Vis,C D, and EPR spectroscopy at asubstoichiometric Cu II ratio.The results indicate...

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Short peptides are not reliable models of thermodynamic and kinetic properties of the N‐terminal metal binding site in serum albumin

Cited by 97 publications

References 42 publications

Thermodynamic study of Cu2+ binding to the DAHK and GHK peptides by isothermal titration calorimetry (ITC) with the weaker competitor glycine

Thermodynamic study of Cu2+ binding to the DAHK and GHK peptides by isothermal titration calorimetry (ITC) with the weaker competitor glycine

Method to Site-Specifically Identify and Quantitate Carbonyl End Products of Protein Oxidation Using Oxidation-Dependent Element Coded Affinity Tags (O-ECAT) and NanoLiquid Chromatography Fourier Transform Mass Spectrometry

A Functional Role for Aβ in Metal Homeostasis? N‐Truncation and High‐Affinity Copper Binding

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