Imidazole Zinc Research Articles

An ultrasensitive stain for negative protein detection in SDS-PAGE via 4′,5′-Dibromofluorescein

A highly sensitive method for brief and economical staining of proteins in 1-D and 2-D sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) by 4′,5′-Dibromofluorescein (DBF) was developed in this study. Down to 0.025–0.05ng protein could be detected within 10min (only 2 steps) by DBF stain, which is approximately 10-fold more sensitive than those of Eosin Y (EY) and SYPRO Ruby stains, and 20-fold more sensitive than that of imidazole-zinc (IZ) negative stain. In addition, the LC-MS/MS results indicated that the newly developed staining method is compatible with the downstream protein identification. Moreover, the mechanism of DBF stain was investigated by molecular docking.

A method for rapid and sensitive negative staining of proteins in SDS-PAGE using 2',7'-dichlorofluorescein.

We report here a rapid and sensitive technique for negative visualization of protein in 1D and 2D SDS-PAGE by using 2', 7'-dichlorofluorescein (DCF), which appeared as transparent and colorless bands in an opaque gel matrix background. For DCF stain, down to 0.1-0.2ng protein could be easily visualized within 7min by only two steps, and the staining is fourfold more sensitive than that of Eosin Y (EY) negative stain and glutaraldehyde (GA) silver stain, and eightfold more sensitive than that of the commonly used imidazole-zinc (IZ) negative stain. Furthermore, DCF stain provided good reproducibility, linearity, and MS compatibility compared with those of IZ stain. In addition, the potential staining mechanism was investigated by colorimetric experiment and molecular docking, and the results demonstrated that the interaction between DCF and protein occurs mainly via van der waals force, electrostatic interaction, and hydrogen bonding.

Lipopolysaccharide aggregates in native agarose gels detected by reversible negative staining with imidazole and zinc salts

We investigated the use of imidazole and zinc salts for the detection of lipopolysaccharide (LPS) aggregates separated by native agarose gel electrophoresis (NAGE). As a result, a new staining procedure was established by which as little as 1.5μg of Escherichia coli O55:B5 LPS aggregates was detected by means of inducing a clear, transparent pattern, contrasted against an opaque background. E. coli O55:B5 LPS preparations treated with nucleases and proteinase K proved that the reverse-stained LPS pattern is not related to any potential artifacts caused by unrelated biomolecules (e.g., nucleic acids, proteins). After this, we showed that the procedure is applicable to two-dimensional LPS separation using NAGE/SDS-PAGE, while at the same time confirming that real polydisperse LPS aggregates are represented by the stained profile. Also, we demonstrated the general applicability of this stain to the detection of different NAGE-separated LPS aggregates (e.g., from E. coli 026:B6, E. coli 0111:B4, Salmonella minnesota Re595). Finally, using lysozyme as a model protein, we found that imidazole–zinc may be combined with Coomassie brilliant blue R-250 into a double-staining process to enable the use of NAGE for investigating the interaction of cationic proteins and LPS aggregates and protein or LPS concentration effects on protein–LPS binding.

Supramolecular Control of Biomimetic Coordination – ZnII Cavity Complexes Presenting Two Differentiated Labile Sites in cis Positions

AbstractA supramolecular approach to model the active sites of metalloenzymes is to associate a cavity with a tripodal coordination core. One key feature of many enzymes is the possible binding of two different ligands in cis positions relative to each other, which has not yet been described for cavity complexes. Here, the bowl shape of resorcinarene allows such a coordination environment for ZnII complexes. A detailed NMR study with various carboxylic acids evidences size‐, shape‐, and, thus, regioselectivity for carboxylate coordination in the endo position, the exo position, or both. The coordination of diketonates unambiguously demonstrates the relative cis position of the two labile sites present in the tris(imidazole)zinc(II) bowl complex. An interesting intramolecular exchange process was also observed. Finally, a comparison with calix[6]arene‐based complexes (so‐called funnel complexes) further highlights the key role of the cavity in the control of properties of the metal ion.

Negative staining of lipopolysaccharides on polyacrylamide gels by using eosin B

A sensitive and simple technique for the negative detection of lipopolysaccharides (LPSs) following polyacrylamide gel electrophoresis (PAGE) using eosin B (EB) was developed. After electrophoresis, gels were fixed, stained, and developed within 30min to achieve transparent and colorless LPS bands under opaque gel matrix background. As low as 20 to 40ng of total LPSs could be detected, which is 4-fold more sensitive than those of the widely used silver stain developed by Fomsgaard and coworkers and imidazole–zinc (IZ) negative stain. For its sensitivity and brevity, this stain may be a practical method for LPS determination in the routine laboratory.

High-Field Solid-State 67Zn NMR Spectroscopy of Several Zinc−Amino Acid Complexes

We report the results of our solid-state (67)Zn NMR study of the various zinc sites in four zinc-amino acid coordination complexes: bis(glycinato)zinc(II) monohydrate; bis(l-alaninato)zinc(II); bis(l-histidinato)zinc(II) dihydrate; and sodium bis(l-cysteinato)zincate(II) hexahydrate; as well as a related complex, bis(imidazole)zinc(II) chloride. We demonstrate the advantages of using high (21.1 T) applied magnetic fields for detecting (67)Zn directly at ambient temperatures using the quadrupolar Carr-Purcell Meiboom-Gill (QCPMG) pulse sequence. The stepped-frequency technique was employed in cases where the central-transition (CT) (67)Zn NMR spectra were too broad to be uniformly excited. The parameters of the anisotropic zinc tensors were extracted by iterative simulations of the experimental spectra. In all cases, the quadrupolar interaction is found to dominate the central-transition (67)Zn NMR spectra; no convincing effects from chemical shift anisotropy (CSA) on the NMR spectra of the five complexes could be reliably detected at this field strength. Analyses of the experimental NMR spectra reveal that the (67)Zn quadrupolar coupling constants (C(Q)) range from 7.05 to 26.4 MHz, the isotropic chemical shifts (delta(iso)) range from 140 to 265 ppm, and the quadrupolar asymmetry parameters (eta(Q)) range from 0.20 to 0.95. The first report of the NMR spectral features of pentacoordinated zinc sites is included for two complexes. Quantum chemical calculations of the electric field gradient (EFG) and magnetic shielding tensors reproduced the experimental results to a reasonable extent. Moreover, the computationally determined orientations of both tensors permit correlations between NMR tensor properties and zinc local environments to be understood.

Studies on the zinc(II)-azoimine system. Single-crystal X-ray structure of Zn(MeaaiMe)Cl 2·H 2O and Zn(HaaiMe) 2(NCS) 2 (MeaaiMe=1-methyl-2-( p-tolylazo)imidazole, HaaiMe=1-methyl-2-(phenylazo) imidazole)

Dichloro zinc(II) complexes of 1-alkyl-2-(arylazo)imidazole, Zn(RaaiR′)Cl 2·H 2O (RaaiR′= p-RC 6H 4N=NC 3H 2NN(1)R′: R=H, Me, Cl and R′=Me, CH 2CH 3, CH 2Ph) are characterized by elemental analysis, IR, UV–Vis and 1H NMR spectral data. The reaction of ZnCl 2 and RaaiR′ (excess) in the presence of NH 4CNS has synthesized Zn(RaaiR′) 2(NCS) 2. The single-crystal X-ray structure of dichloro-{1-methyl-2-( p-tolylazo)imidazole}zinc(II)-monohydrate (Zn(MeaaiMe)Cl 2·H 2O) suggests that the complex is distorted trigonal bipyramidal (TBP) around Zn(II) with a trigonal plane constituted from Cl(1), Cl(2), N(imidazole) (N(1)). Molecular packing shows a 1D chain via a hydrogen bonded eight membered ring. The X-ray structure of di-thiocyanato-bis-{1-methyl-2-(phenylazo)-imidazole}zinc(II) also shows a distorted TBP around Zn(II). The coordination sphere is ZnN 3 type: where one of the ligands acts as a bidentate chelating agent and the second one coordinates via only the imidazole-N along with two N-centers from the NCS group.

A model complex for carbonic anhydrases: Crystal structure of tris-imidazole maleato zinc(II) dihydrate

A novel zinc complex containing three simple imidazole rings as a model compound for carbonic anhydrase and as a rare model for the carboxylate–imidazole–zinc triad interactions frequently observed in zinc enzymes has been made and its structure determined by X-ray diffraction in which the zinc atom has a distorted tetrahedral geometry.