Dendrimers are extensively studied for drug delivery and catalysis, most of which are pH dependent. Phosphate buffer solutions (PBSs) are often used to adjust the pH. We have found that phosphate ions become incorporated into poly(amidoamine) (PAMAM) dendrimer molecules by forming H-bonds with tertiary nitrogens. We show that this specific interaction between H2PO4- and HPO42- ions and generation five PAMAM dendrimers causes a decrease in hydrodynamic size, disturbing the outcome of the size exclusion chromatography analysis. We monitored this interaction by 1H and 31P high resolution NMR, NMR-diffusiometry, pH-potentiometry and infrared spectroscopy. Failing to take into account this effect may lead to incorrect conclusions and misinterpreting interactions of PAMAM dendrimers with drug molecules and subsequently incorrect dosing. The phosphate salts of amino terminated generation five PAMAM dendrimers are stable for years when stored in the dark, even in dilute aqueous solutions, which has important implications for the shelf-life of dendrimer-based drug delivery systems.

Solvent self-diffusion coefficients D*(w) and D-w are respectively determined by PGSE NMR in the bulk solvent and in suitably prepared aqueous micellar solutions of a series of commercial ethoxylated nonyl phenols (ENPs) at 298 and 317 K. The capability of ENP micelles for obstructing the free diffusion of solvent molecules is characterized by the obstruction ratio K = D-w/D*(w), which turns out to depend merely on the volume fraction of EO units present-regardless of significant changes in solute concentration, distribution of EO chain length, micellar aggregation number and temperature. The experiments are interpreted in terms of a renewed analytical model of ENP micellar structure, based on Tanford's concept of hydrodynamic particle. The results from model fitting return the characteristic sizes of ENP monomers, support the ellipsoid shape of the micelles, indicate a stable EO shell, follow the trend in the variation of hydration number vs. the chain length predicted by thermal analysis in PEO solutions.

In order to model the biotransformation reactions of half-sandwich type platinum metal complexes with potential antiproliferative activity in the serum the interaction between [(eta(6)-p-cym)Ru(H2O)(3)](2+) and N-methylimidazole was studied in aqueous 0.20 M KNO3 or KCI medium with the combined use of pH-potentiometric, NMR, UV Vis and ESI-TOF-MS techniques. The results indicate that up to three ligands can coordinate to the metal ion in rather slow processes resulting in mononuclear species. In the absence of chloride ions various mixed hydroxido complexes are also identified under basic conditions. Chloride was found to take the free coordination site(s) of the metal ion in the 1:1 and 1:2 species and to hinder effectively the hydrolytic processes. Stability constants of the species are reported. The results indicate that even this monodentate model ligand, N-methylimidazole, forms stable complexes with the metal ion under physiologically relevant conditions at 1:3 metal to ligand ratio and prevents it from complete hydrolysis at pH = 7.4. (C) 2015 Elsevier BY. All rights reserved.

Oxidative stress (OS) is common to numerous diseases, including cancer. Among other, OS is mediated by the activity of superoxide dismutases (SOD), natural proteins that catalyze dismutation of the superoxide anion. SOD-activity can efficiently be mimicked by various compounds including Mn porphyrins. A thorough study of protolytic speciation of manganese ortho- (MnTE-2-PyP) and meta-tetrakis(N-ethylpyridinium-3-yl)porphyrin (MnTE-3-PyP) complexes revealed that in aqueous solutions within the pH range 2–13 the complexes could be mono- and di-deprotonated. The rate constants of the water-exchange reactions of MnIIITE-2-PyP5+ in slightly acidic aqueous medium (pH = 6.0) have also been reported along with the corresponding activation parameters. In highly basic solutions (pH = 12) MnIIITE-2-PyP5+ complex shown no measurable relaxivity enhancement indicating that dominant porphyrin species at this pH must be the one which coordinates neither the aqua nor hydroxo ligand, i.e. in which axially coordinated to the central Mn(III) is only an oxo ligand . As a continuation of our previous studies we present a systematic 17O-NMR kinetic study of pH-dependence of the water exchange of MnIIITE-2-PyP5+ and MnIIITE-3-PyP5+ complexes in the pH range 2-13.

Medium generation PAMAM dendrimers are extensively researched as drug delivery vehicles therefore detailed knowledge of their physico-chemical properties in solution is vital. We have selected ethylenediamine core, generation five poly(amidoamine) (PAMAM_E5) dendrimers (amine terminated PAMAM_E5.NH2 and its succinamic acid derivative PAMAM_E5.SAH, respectively) as model compounds to study their dynamic behavior in water as a function of pH and concentration. Diffusion coefficients of water and the dendrimers were determined in deuterium oxide using pulse-field gradient stimulated echo (PGSE) NMR. The diffusion rate for PAMAM_E5.NH2 increased monotonously with increasing pH values while for PAMAM_E5.SAH a maximum was found at an isoelectric value of pH = 5.7. The apparent diffusion coefficients of dendrimers decreased linearly with increasing concentration measured at their self-pH (pH = 9.4 for PAMAM_E5.NH2 and pH = 5.7 for PAMAM_E5.SAH) in the absence of added salts. The observations could be explained by considering hard sphere interactions between strongly hydrated dendrimer molecules. The average hydrodynamic radii of dendrimers were determined by extrapolating the measured diffusion coefficients to zero dendrimer concentration and applying the Stokes–Einstein equation. The calculated values were RH = 3.05 ± 0.04 nm for PAMAM_E5.NH2 and RH = 3.37 ± 0.08 nm for PAMAM_E5.SAH respectively. Measured diffusion coefficients of water (D2O) also decreased linearly with increasing dendrimer concentration. From the concentration dependence, the average number of water molecules that form one dynamic unit with one macromolecule could be calculated on the basis of three different obstruction models. It has been concluded that hydrated PAMAM dendrimers in aqueous solutions behave as soft colloids against solvent molecules but as hard-sphere colloids against each other. Their equivalent hard-sphere radii were found to be equal to the measured hydrodynamic radii.

The tetrapeptides Ac-SKHM-NH2, Ac-TKHM-NH2, Ac-MKHS-NH2, Ac-S(OMe)KHM-NH2, and Ac-MKHS(OMe)-NH2 and the nonapeptides Ac-KTNSKHMAG-NH2 and Ac-KTNMKHSAG-NH2 were synthesized and their copper(II) complexes were studied by potentiometric, UV–Vis, circular dichroism (CD), and electron paramagnetic resonance (EPR) spectroscopic methods. These peptides mimic the 109–112 and 106–114 residues of the sequence of human prion protein. The imidazole-N donor atoms of histidyl residues were found to be the primary metal binding sites of all peptide fragments. This binding mode provides a good possibility for the cooperative deprotonation and metal ion coordination of two amide functions preceding histidine. The (Nim,N−,N−)-bonded species predominate in the pH range 5.5–7.0 and the free coordination sites of these species make possible the metal binding of weakly coordinating side chains. The comparison of the potentiometric and spectroscopic results revealed the stabilizing role of the oxygen donors of seryl, threonyl, or methoxyseryl residues of Ac-SKHM-NH2, Ac-TKHM-NH2, Ac-S(OMe)KHM-NH2, and Ac-KTNSKHMAG-NH2 containing the mutations in position 109. These interactions were, however, not observed in the peptides containing the specific amino acids in other locations of the peptide sequence.

The formation of mixed copper(II) and zinc(II) complexes with Aβ(1-16)-PEG has been investigated. The peptide fragment forms stable mixed metal complexes at physiological pH in which the His13/His14 dyad is the zinc(II)'s preferred binding site, while copper(II) coordination occurs at the N-terminus also involving the His6 imidazole. Copper(II) is prevented by zinc(II) excess from the binding to the two His residues, His13 and His14. As the latter binding mode has been recently invoked to explain the redox activity of the copper-Aβ complex, the formation of ternary metal complexes may justify the recently proposed protective role of zinc(II) in Alzheimer's disease. Therefore, the reported results suggest that zinc(II) competes with copper for Aβ binding and inhibits copper-mediated Aβ redox chemistry.

Aggregation of the amyloid beta-peptide (Abeta) into insoluble fibrils is a key pathological event in Alzheimer's Disease (AD). There is now compelling evidence that metal binding to Abeta is involved in AD pathogenesis. The amino acid region 1-16 is widely considered as the metal binding domain of Abeta. In this work, we used a combined potentiometric, NMR, and electrospray ionization mass spectrometry (ESI-MS) approach to study the zinc(II) binding to a new polyethylene glycol (PEG)-conjugated peptide fragment encompassing the 1-16 amino acid sequence of Abeta (Abeta(1-16)PEG). Our results demonstrate for the first time that the Abeta(1-16) is able to coordinate up to three zinc ions, all the histidyl residues acting as independent anchor sites. The study was complemented by systematically investigating the zinc(II) complexes of a series of shorter peptide fragments related to the Abeta(1-16) sequence, namely, Abeta(1-4), Abeta(1-6), AcAbeta(1-6), AcAbeta(8-16)Y10A. The comparison of the whole results allowed the identification of the zinc(II) preferred binding sites within the longer Abeta(1-16) amino acid sequence. Unlike copper(II) that prefers the N-terminal amino group as the main binding site, the zinc(II) is preferentially placed in the 8-16 amino acidic region of Abeta(1-16).

1. Multihisztidin peptidek rez(II)- es cink(II)komplexei: A hisztidin nitrogen donoratomok a peptidek elsődleges femkotőhelyei. Ezek koordinaciojaval makrokelatok kepződhetnek, amelyek stabilitasa a hisztidinek szamatol es tavolsagatol fugg. A karboxilcsoportok jelenlete a cink(II)komplexek stabilitasat noveli. A rez(II)ionok az amidcsoport deprotonalodasat is indukalhatjak, ami tobbmagvu komplexek kepződesehez vezet. A megkotott rezionok szama megegyezik a hisztidinek szamaval. 2. A prion protein peptid fragmenseinek femkomplexei: Az oktarepeaten kivuli hisztidinek is stabilis rezkotőhelyek. A HuPrP(84-114) fragmensre kapott eredmenyek szerint a kotesi helyek stabilitasi sora: His111 > His96 >> His85. Egyeb atmenetifemek komplexeit is tanulmanyoztuk, amelyek stabilitasi sora a kovetkező: Pd(II) > Cu(II) > Ni(II) > Zn(II) > Cd(II) ~ Co(II) > Mn(II). 3. Az amyloid-? peptid rez(II)komplexei: Az A?(1-16) peptidnek kiugroan nagy rezionaffinitasa van. A terminalis aminocsoport az elsődleges femkotőhely, amit a hisztidinek koordinacioja kovet. Egy A?(1-16) molekula 4 reziont kepes megkotni. Az egy- ket- es harom-magvu komplexeknek koordinacios izomerjei lehetnek, de a terminalis aminocsoport es a szomszedos amidnitrogenek koordinacioja preferalt. | 1. Copper(II) and zinc(II) complexes of multihistidine peptides: Histidyl residues are the primary metal binding sites resulting in the formation of macrochelates. The stabilities of macrochelates are influenced by the number and location of histidyl residues. The stability of zinc(II) complexes is enhanced by the presence of carboxylate functions. Formation of polynuclear complexes has also been detected and their nuclearities correspond to the number of histidyl sites. 2. Metal binding affinity of prion peptide fragments: Histidyl residues outside the octarerepat domain are effective copper binding sites. The results obtained for the copper(II) complexes of HuPrP(84-114) revealed the following stability order: His111 > His96 >> His85. Complex formation with several other transition elements has also been studied and their stability order: Pd(II) > Cu(II) > Ni(II) > Zn(II) > Cd(II) ~ Co(II) > Mn(II). 3. Copper(II) complexes of amyloid-? peptide fragments: A?(1-16) has an outstanding affinity towards the complexation with copper. The terminal amino group is the primary metal binding site, followed by the coordination of histidyl residues. One molecule of A?(1-16) can bind as much as four copper(II) ions. Various coordination isomers of the mono-, di- and tri-nuclear complexes can exist with a preference for the coordination via the terminal amino and subsequent amide groups.

Complex formation processes between the 39-mer residue peptide fragment of human prion protein, HuPrP(76-114), and copper(II) ions have been studied by potentiometric, UV-vis, circular dichroism (CD), electron paramagnetic resonance, and electrospray ionization mass spectrometry methods. This peptide consists of 39 amino acid residues and contains two histidines (His77 and His85) belonging to the octarepeat domain and two histidines (His96 and His111) outside this domain. It was found that HuPrP(76-114) is able to bind 4 equiv of metal ions and all histidyl residues are independent, except nonequivalent metal binding sites in the oligonuclear species. Imidazole nitrogen donor atoms are the primary and exclusive metal binding sites below pH 5.5 in the form of various macrochelates. The macrochelation slightly suppresses, but cannot prevent, the deprotonation and metal ion coordination of amide functions, resulting in the formation of (N-im,N-), (N-im, N-,N-), and (N-im, N-, N-, N-)-coordinated copper(II) complexes in the pH range from 5.5 to 9. CD spectroscopy results gave clear evidence for the differences in the metal binding affinity of the histidyl sites according to the following order: His111 > His96 >> His77 similar to His85. Among the oligonuclear complexes, the formation of di- and tetranuclear species seems to be favored over the trinuclear ones, at pH values beyond the physiological ones. This phenomenon was not observed in the complex formation reactions of HuPrP(84-114), a peptide fragment containing only one histidyl residue from the octarepeat. As a consequence, the data support the existence of cooperativity in the metal binding ability of this peptide probably due to the presence of two octarepeat sequences of the dimeric octarepeat domain of HuPrP(76-114) at basic pH values.

The solution conformation and the copper(II) binding properties have comparatively been investigated for the two novel hexapeptides Ac-HPSGHA-NH2 (P2) and Ac-HGSPHA-NH2 (P4). The study has been carried out by means of CD, NMR, EPR and UV-Vis spectroscopic techniques in addition to potentiometric measurements to determine the stability constants of the different copper(II) complex species formed in the pH range 3–11. The peptides contain two histidine residues as anchor sites for the metal ion and differ only for the exchanged position of the proline residue with glycine. CD and NMR results for the uncomplexed peptide ligands suggest a predominantly unstructured peptide chain in aqueous solution. Potentiometric and spectroscopic data (UV-Vis, CD and EPR) show that both peptides strongly interact with copper(II) ions by forming complexes with identical stoichiometries but different structures. Furthermore, Far-UV CD experiments indicate that the conformation of the peptides is dramatically affected following copper(II) complexation with the P4 peptide adopting a β-turn-like conformation.

Alzheimer's disease (AD) is becoming a rapidly growing health problem, as it is one of the main causes of dementia in the elderly. Interestingly, copper(II) (together with zinc and iron) ions are accumulated in amyloid deposits, suggesting that metal binding to A beta could be involved in AD pathogenesis. In A beta, the metal binding is believed to occur within the N-terminal region encompassing the amino acid residues 1-16. In this work, potentiometric, spectroscopic (UV-vis, circular dichroism, and electron paramagnetic resonance), and electrospray ionization mass spectrometry (ESI-MS) approaches were used to investigate the copper(II) coordination features of a new polyethylene glycol (PEG)-conjugated A beta peptide fragment encompassing the 1-16 amino acid residues of the N-terminal region (A beta(1 - 16)PEG). The high water solubility of the resulting metal complexes allowed us to obtain a complete complex speciation at different metal-to-ligand ratios ranging from 1:1 to 4:1. Potentiometric and ESI-MS data indicate that A beta(1-16)PEG is able to bind up to four copper(II) ions. Furthermore, in order to establish the coordination environment at each metal binding site, a series of shorter peptide fragments of A beta, namely, A beta(1-4), A beta(1-6), AcA beta(1-6), and AcA beta(8-16)Y10A, were synthesized, each encompassing a potential copper(II) binding site. The complexation properties of these shorter peptides were also comparatively investigated by using the same experimental approach.

Tissue transglutaminase (TG2) catalyzes the Ca(2+)-dependent posttranslational modification of proteins via formation of isopeptide bonds between their glutamine and lysine residues. Although substrate specificity of TG2 has been studied repeatedly at the sequence level, no clear consensus sequences have been determined so far. With the use of the extensive structural information on TG2 substrate proteins listed in TRANSDAB Wiki database, a slight preference of TG2 for glutamine and lysine residues situated in turns could be observed. When the spatial environment of the favored glutamine and lysine residues was analyzed with logistic regression, the presence of specific amino acid patterns was identified. By using the occurrence of the predictor amino acids as selection criteria, several polypeptides were predicted and later identified as novel in vitro substrates for TG2. By studying the sequence of TG2 substrate proteins lacking available crystal structure, the strong favorable influence on substrate selection of the presence of substrate glutamine and lysine residues in intrinsically disordered regions could also be revealed. The collected structural data have provided novel understanding of how this versatile enzyme selects its substrates in various cell compartments and tissues.

Terminally protected pentapeptides with 2 histidines (Ac-HHVGD-NH2 and Ac-HVGDH-NH2) and the terminally free peptides containing both internal aspartyl and C-terminal histidyl residues (FDAH and VIDAH) have been synthesized, and copper(II) complexes studied by potentiometric, UV-Vis, CD, and EPR spectroscopic techniques in solution. Both thermodynamic and spectroscopic data reveal that side chain donor atoms of aspartyl and histidyl residues have a significant contribution to the metal binding affinity of peptide molecules. In the case of terminally protected peptides, the role of the imidazole-N donor functions is reflected in the enhanced stability of the 3N and 4N coordinated copper(II) complexes. The amino and β-carboxylate groups of FDAH and VIDAH create a very effective metal binding site with the (NH2, N−, β-COO−) and (NH2, N−, N−, β-COO−) coordination modes including the N-termini, while the histidine sites are available for the formation of the (Nim, N−, N−) binding mode resulting in the preference of dinuclear complex formation.

A 31-mer polypeptide, which encompasses residues 84-114 of human prion protein HuPrP(84-114) and contains three histidyl residues, namely one from the octarepeat (His85) and two histidyl residues from outside the octarepeat region (His96 and His111), and its mutants with two histidyl residues HuPrP(84-114)His85Ala, HuPrP(84-114) His96Ala, HuPrP(84-114)His111Ala and HuPrP(91-115) have been synthesised and their Cu2+ complexes studied by potentiometric and spectroscopic (UV/Vis, CD, EPR, ESI-MS) techniques. The results revealed a high Cu2+-binding affinity of all peptides, and the spectroscopic studies made it possible to clarify the coordination mode of the peptides in the different complex species. The imidazole nitrogen donor atoms of histidyl residues are the exclusive metal-binding sites below pH 5.5, and they have a preference for macrochelate structure formation. The deprotonation and metal-ion coordination of amide functions take place by increasing the pH; all of the histidines can be considered to be independent metal-binding sites in these species. As a consequence, di- and trinuclear complexes can be present even in equimolar samples of the metal ion and peptides, but the ratios of polynuclear species do not exceed the statistically expected ones; this excludes the possibility of cooperative Cu2+ binding. The species with a (N(im),N,N)-binding mode are favoured around pH 7, and their stability is enhanced by the macrochelation from another histidyl residue in the mononuclear complexes. The independence of the histidyl sites results in the existence of coordination isomers and the preference for metal binding follows the order of: His111>His96>His85. Deprotonation and metal-ion coordination of the third amide functions were detected in slightly alkaline solutions at each of the metal-binding sites; all had a (N(im),N,N,N)-coordination mode. Spectroscopic measurements also made it clear that the four lysyl amino groups of the peptides are not metal-binding sites in any cases.