Design of thiolate rich metal binding sites within a peptidic framework

A de novo protein design strategy provides a powerful tool to elucidate how heavy metals interact with proteins.Cysteine derivatives of the TRI peptide family (Ac-G(LKALEEK)4G-NH2) have been shown to bind heavy metals in an unusual trigonal geometry. Our present objective was to design binding sites in R-helical scaffolds that are able to form higher coordination number complexes with Cd(II) and Hg(II). Herein, we evaluate the binding of Cd(II) and Hg(II) to double cysteine substituted TRI peptides lacking intervening leucines between sulfurs in the heptads. We compare a -Cysd-X-X-X-Cysa- binding motif found in TRIL12CL16C to the more common -Cysa-X-X-Cysd- sequence of native proteins found in TRIL9CL12C. Compared to TRI, these substitutions destabilize the helical aggregates,leading to mixtures of two- and three-stranded bundles. The three-stranded coiled coils are stabilized by the addition of metals. TRIL9CL12C forms distorted tetrahedral complexes with both Cd(II) and Hg(II), as supported by UV-vis,CD, 113Cd NMR, 199Hg NMR and 111mCd PAC spectroscopy. Additionally, these signatures are very similar to those found for heavy metal substituted rubredoxin. These results suggest that in terms of Hg(II) binding, TRIL9CL12Ccan be considered as a good mimic of the metallochaperone HAH1, that has previously been shown to form protein dimers. TRIL12CL16C has limited ability to generate homoleptic tetrahedral complexes (Cd(SR)42-). These type of complexes were identified only for Hg(II). However, the spectroscopic signatures suggest a different geometry around the metal ion, demonstrating that effective metal sequestration into the hydrophobic interior of the bundle requires more than simply adding two sulfur residues in adjacent layers of the peptide core. Thus, proper design of metal binding sites must also consider the orientation of cysteine sidechains in a vs d positions of the heptads.     

Synthesis and characterization of a new fluorene ligand and its complexes with cobalt(II), copper(II), and ruthenium(II)

A new fluorene ligand, benzo[15-crown-5]-5H-pyrido[3′,2′:4,5]cylopenta[1,2-b]pyridin-5-ylidenehydrazone (bph), has been synthesized from the reaction of 4,5-diazafluoren-9-one with 4′-formylbenzo-15-crown-5. The Co(II), Cu(II), and Ru(II) complexes of the ligand were prepared and characterized. The metal-to-ligand ratio of the Co(II) and Cu(II) complexes was found to be 2:1 and that of the Ru(II) complex was found to be 1:1. The ligand and complexes have been characterized by FTIR, UV–visible, ¹H NMR and fluorescence spectra, as well as elemental analyses and mass spectra.     

Synthesis of 4,5,6,7‐Tetrabromo‐1H‐benzimidazole Derivatives

A convenient and simple method for the preparation of polybrominated benzimidazole derivatives has been described. Reaction of 4,5,6,7‐tetrabromo‐1‐(3‐chloropropyl)‐1H‐benzimidazole, obtained by alkylation of 4,5,6,7‐tetrabromo‐1H‐benzimidazole (TBBi) with 1‐bromo‐3‐chloropropane in the presence of KOH, with morpholine, aniline, benzylamine, N‐methyl piperazine, and 2‐arylpyrrolidines afforded new TBBi derivatives.     

Review: Recent advances in the investigations of NO activation on cobalt and manganese porphyrins: a brief review

Cobalt and manganese porphyrins are known for their ability to activate small molecules. This is particularly important in the case of nitric oxide, whose role and mechanism of action in the redox biological systems have not yet been fully recognized. The goal of this article is to draw attention to some of the current trends of research in this area. The interactions involving NO itself and the primary products of its oxidation (nitrite) and reduction (nitroxyl) have been distinguished and separately discussed. The diversity of undertaken issues sheds light on both the expected behavior in biologically relevant systems as well as potential practical applications.     

Comparison of mouse plasma and brain tissue homogenate sample pretreatment methods prior to high‐performance liquid chromatography for a new 1,2,4‐triazole derivative with anticonvulsant activity

The focus of the study was to develop a bio‐analytical assay for a 1,2,4‐triazole derivative from plasma and brain tissue homogenate samples. The goal was to compare analytical techniques that facilitate high accuracy with simplified sample processing. In this study, commonly used standard protein precipitation and solid‐phase extraction methods utilizing C₁₈ and cartridges of Hybrid technology were compared in terms of their ability for sample pretreatment and removal of biological matrices before high‐performance liquid chromatography quantification. Fast classical reversed‐phase chromatography on a C₁₈ column paired with selective sample preparation using Hybrid solid‐phase extraction technology resulted in the most precise bio‐analytical determination of the hydrophobic 1,2,4‐triazole derivative in both biological samples studied. The obtained recovery values were above 95% with the coefficient of variation lower than 5%.     

Search for γ-Emission from Isomeric Nuclei Induced by MeV Electron Beams

In this paper we study the nuclear transitions induced by incident electrons having an energy of several MeV's. We measured the cross sections for the excitation of isomeric nuclear states by 7.7 MeV electrons. The cross sections were 1.2 μb for ^111m Cd, 5.5 μb for ^113m In and 7.0 μb for ^115m In. The peak activation rates were 1.8×10⁻¹² s⁻¹ for ^111m Cd, 8.1×10⁻¹² s⁻¹ for ^113m In and 1.0×10⁻¹¹ s⁻¹ for ^115m In, for a peak power of the electron beam of 1.8×10⁶ W cm⁻². Then we describe for the first time the results of a series of experiments in which samples containing the isomeric nuclei ^166m Ho and ^186m Re have been irradiated with MeV electron beams. An upper limit of 17 mb has been determined for the cross section of electron-induced γ-emission from ^166m Ho and an upper limit of 2.2 mb has been determined for the cross section of electron-induced γ-emission from ^186m Re. This revised version was published online in July 2006 with corrections to the Cover Date.     

Effect of Calcium Compound Type and Dosage on the Properties of Acid Rennet Goat’s Milk Gels

The aim of this study was to determine the effect of adding calcium compounds to processed goat’s milk, and on the properties of acid rennet goat’s milk gels, which are a middle product obtained in the manufacture of acid rennet cheese. The properties of the gels directly affect the quality of acid rennet cheeses. The analysis of raw goat’s milk was carried out, then acid rennet gels were produced with the addition of six different calcium compounds (chloride, citrate, bisglycinate, gluconate, lactate, and carbonate). The dynamics of milk fermentation were performed by monitoring the pH value of milk during acidification. The pH, syneresis, color, and texture profile were determined in the formulated acid rennet gels. An organoleptic evaluation was also performed. The study demonstrated that, not only calcium chloride, but also calcium citrate, gluconate, lactate, bisglycinate, and calcium carbonate could be used in the production of goat’s milk acid rennet gels, or the middle product in the manufacture of acid rennet curd cheese from goat’s milk. Notably, the addition of citrate, bisglycinate, and calcium carbonate in doses of 20 mg Ca 100 g⁻¹ most effectively reduced syneresis compared to the control sample by 4.76% (citrate), 7.85% (bisglycinate), and 10.28% (carbonate). The hardness of the control gels ranged from 2.35 N to 2.99 N. The addition of chloride, citrate, gluconate, lactate, and calcium carbonate to the milk improved the acid rennet gel’s hardness. The addition of 20 mg Ca 100 g⁻¹ as gluconate increased the hardness the most (3.61 N). When increasing the calcium dosage in the form of all compounds, there was a tendency to increase the gel’s springiness. The addition of chloride, citrate, and bisglycinate to milk did not result in a darkening of the gel’s color. The addition of calcium compounds mostly reduced the intensity of goatish taste and odor. Calcium gluconate, in particular, reduced the goatish taste the most, a taste which is not always acceptable by the consumers.