Tris-HCl Buffer pH Research Articles

Process Development for the Production and Purification of Adeno-Associated Virus (AAV)2 Vector using Baculovirus-Insect Cell Culture System.

Adeno-associated viruses (AAV) are promising vectors for gene therapy applications. Here, the AAV2 vector is produced by co-culture of Spodoptera frugiperda (Sf9) cells with Sf9 cells infected with baculovirus (BV)-AAV2-GFP (or therapeutic gene) and BV-AAV2-rep-cap in serum-free suspension culture. Cells are cultured in a flask in an orbital shaker or Wave bioreactor. To release the AAV particles, producer cells are lysed in buffer containing detergent, which is subsequently clarified by low-speed centrifugation and filtration. AAV particles are purified from the cell lysate using AVB Sepharose column chromatography, which binds AAV particles. Bound particles are washed with PBS to remove contaminants and eluted from the resin using sodium citrate buffer at pH 3.0. The acidic eluate is neutralized with alkaline Tris-HCl buffer (pH 8.0), diluted with phosphate-buffered saline (PBS), and further concentrated with tangential flow filtration (TFF). The protocol describes small-scale pre-clinical vector production compatible with scale-up to large-scale clinical-grade AAV manufacturing for human gene therapy applications.

Serum Phenoloxidase Activity in the Hemolymph of Giant Freshwater Prawn Macrobrachium rosenbergii (De Man, 1879)

Melanization is one of the major immune responses in arthropods, particularly crustaceans. The prophenoloxidase (proPO) catalyzes the oxidation of mono or di or polyphenols, a reaction that is the key initial step of melanin formation. We attempted to study the immune response in M. rosenbergii by melanization reaction as produced by serum phenoloxidase (PO) activity. The substrate affinity of the PO enzyme was determined using different phenolic substrates and it was found that the mono, di and poly phenols were oxidized hence the enzyme was characterized as catecholoxidase and l-3,4-dihydroxyphenylalanine (L–DOPA) showed the highest substrate affinity to the enzyme. The biochemical parameters that determined optimum enzyme activity were found such as 1 mM L–DOPA concentration showing at the absorbance of 470 nm, 10 mM Tris–HCl buffer pH 7.5 (brown colour formation), temperature 40 ° C and 10 min incubation. Kinetic characteristics of serum from the prawn were also determined. Determination of optimal conditions of PO activity in the serum has also been attempted. We also identified the serum having a potent PO activity was inhibited by phenylthiourea (PTU) and activation of such as trypsin, sodium dodecyl sulphate (SDS) and laminarin in the freshwater prawn M. rosenbergii.

Sequence Analysis and Preliminary X-ray Crystallographic Analysis of an Acetylesterase (LgEstI) from Lactococcus garvieae

A gene encoding LgEstI was cloned from a bacterial fish pathogen, Lactococcus garvieae. Sequence and bioinformatic analysis revealed that LgEstI is close to the acetyl esterase family and had maximum similarity to a hydrolase (UniProt: Q5UQ83) from Acanthamoeba polyphaga mimivirus (APMV). Here, we present the results of LgEstI overexpression and purification, and its preliminary X-ray crystallographic analysis. The wild-type LgEstI protein was overexpressed in Escherichia coli, and its enzymatic activity was tested using p-nitrophenyl of varying lengths. LgEstI protein exhibited higher esterase activity toward p-nitrophenyl acetate. To better understand the mechanism underlying LgEstI activity and subject it to protein engineering, we determined the high-resolution crystal structure of LgEstI. First, the wild-type LgEstI protein was crystallized in 0.1 M Tris-HCl buffer (pH 7.1), 0.2 M calcium acetate hydrate, and 19% (w/v) PEG 3000, and the native X-ray diffraction dataset was collected up to 2.0 Å resolution. The crystal structure was successfully determined using a molecular replacement method, and structure refinement and model building are underway. The upcoming complete structural information of LgEstI may elucidate the substrate-binding mechanism and provide novel strategies for subjecting LgEstI to protein engineering.

A novel graphene quantum dots/choline chloride/gold nanoparticles-modified carbon fiber microelectrode for sensitive and selective determination of dopamine in the presence of a high concentration of ascorbic acid

We prepared a three-layer composite of gold nanoparticles (AuNPs), choline (Ch) and graphene quantum dots (GQDs), and applied it to modify a carbon fiber microelectrode (CFME) by potentiostatic deposition to obtain a GQDs/Ch/AuNPs/CFME for high-sensitivity determination of dopamine (DA) without inference from ascorbic acid (AA). The surface morphology of electrodes was investigated by scanning electron microscopy. The electrochemical behavior of DA and AA at different modified electrodes was investigated by cyclic voltammetry and differential pulse voltammetry in Tris-HCl buffer (pH = 9.0). Owing to a synergistic effect, the GQDs/Ch/AuNPs/CFME exhibited an obvious electrocatalytic effect on positively charged DA and a significant shielding effect on negatively charged AA. The influence of experimental variables (duration of modification, pH of the working solution, scan rate) was studied. Under optimum conditions, in the presence of AA (1.0 × 10−3 mol/L), the GQDs/Ch/AuNPs/CFME described a fine linear enhancement for DA oxidation in the concentration range 0.01–10.0 μM with an estimated lower limit of detection of 1.616 nM and limit of quantification of 5.389 nM. The modified electrochemical sensor was applied for DA determination in the presence of high concentration of AA in mouse serum without separation steps and with good recovery.

Streptomyces sp. K47 alkaline proteases: partial purification and analysis by zymography

Microbial enzymes are used as organic catalysts in different industrial processes. In this study, we aimed to produce and investigate alkaline proteases from a novel actinobacterium strain isolated from a Black Sea marine sediment. The optimal production conditions for Streptomyces sp. K47 alkaline proteases was 4-days incubation at 28ºC in a salt-free medium buffered with 50 mM Tris-HCl buffer (pH 9.0) and containing glucose (1.0%, w v-1) and yeast extract (0.5%, w v-1). The enzyme solution was partially purified using (NH4)2SO4 precipitation (40-70%). After desalting, it was purified 1-84 fold with a recovery of 19.42%. Zymogram analyses revealed the presence of more than one protease enzyme. The enzyme solution exhibited maximum activity at pH 9.0 and 37ºC, remaining stable after a 2-hour incubation at all tested conditions. Streptomyces sp. K47 has the potential to be used in industrial processes because of its ability to produce multiple protease enzymes displaying stability in a broad pH and temperature range.

Enhanced degradation of 4-aminobenzenesulfonate by a co-culture of Afipia sp. 624S and Diaphorobacter sp. 624L

Two strains, Afipia sp. 624S and Diaphorobacter sp. 624L, were isolated from an enrichment culture with 4-aminobenzenesulfonate (4-ABS) as the only carbon source. Strain 624S utilized 4-ABS as the only source of carbon and energy and degraded 3.8mM 4-ABS in 2 weeks, releasing a small amount of sulfate ions. On the other hand, strain 624L did not utilize 4-ABS. Additionally, a co-culture of strains 624S and 624L resulted in the enhanced degradation of 4-ABS, and no sulfite was accumulated in the degradation of 4-ABS. When incubated in 50mM Tris-HCl buffer (pH 8.0) containing 2.2mM sodium sulfite, strain 624S exhibited no sulfite oxidation; however, strain 624L completely oxidized the sulfite after 2 days. Furthermore, when manganase, which has the ability to oxidize sulfite, was added to the medium, the degradation rate of 4-ABS was increased in comparison with the non-addition control. These results indicate that the sulfite oxidation might stimulate the degradation of 4-ABS by strain 624S, suggesting syntrophic interaction between strains 624S and 624L based on sulfite oxidation.

Purification of high molecular weight thermotolerant esterase from Serratia sp. and its characterization.

In the present study, an extracellular esterase from Serratia sp. was purified 24.46 fold using an initial ammonium sulphate precipitation step (optimized concentration of 30-40%), followed by Diethylaminoethyl cellulose (DEAE-cellulose) chromatography and size exclusion Sephadex G-200 column chromatography steps. The molecular weight of the esterase using native polyacrylamide gel electrophoresis (PAGE) was determined to be 236kDa and by using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) was found to be 60kDa suggesting that the enzyme was a tetramer of 4 subunits. The purified esterase was able to catalyze the hydrolysis of p-nitrophenyl esters, especially p-nitrophenyl acetate. Maximum esterase activity was achieved in 0.15M Tris-HCl buffer of pH 8.5 at 50°C after 10min. The enzyme was stable for at least 8h at 4 and 35°C but the half-life was determined to be 4.5h at 50°C and 3h at 60°C. The esterase activity was inhibited by detergents (1mM) (Triton X-100, Tween 60, Tween 80, ethylenediamine tetraacetic acid and SDS) except Tween 20. The esterase activity was inhibited by organic solvents (1mM) such as ethanol, methanol, acetone, acetonitrile and was stable in the presence of glycerol, isopropanol but the organic solvent dimethyl sulfoxide (DMSO) significantly (p < 0.05) enhanced esterase activity. The matrix-assisted laser desorption ionization-time of flight mass spectrometry showed that the enzyme exhibited similarity with the pimeloyl-[acyl carrier protein] methyl ester esterase of Serratia marcescens.

Effect of some amino acids on the binding of quercetin and rutin flavonoids with DNA

The fluorescence spectra in combination with UV–Vis absorption were used in the present work. The binding of quercetin and rutin flavonoids with DNA was investigated in Tris-HCl buffer (pH = 7) by using acridine orange dye. A strong fluorescence quenching reaction of DNA-acridine orange dye to these flavonoids was observed where the dye has an emission band at 528 nm after excitation at 490 nm. The binding constant was determined to be 2.99 × 107, 4.14 × 102 mol−1L in case of quercetin and rutin respectively. The type of interaction was determined to be electrostatic for quercetin and hydrophobic for rutin. The binding constant of arginine, asparagine and phenylalanine amino acids with DNA was determined to be 1.8 × 102, 8.71 × 103 and 3.61 × 102 mol−1L respectively. The interaction of quercetin and rutin with DNA–amino acid complexes was studied where the results indicate that binding of these amino acids with DNA has significant effect on increasing the binding constant of the studied flavonoids with DNA, asparagine and phenylalanine has an effect on converting the type of interaction of quercetin with DNA from electrostatic to van der Waals force or hydrogen bond, and arginine convert the interaction type of rutin with DNA from hydrophobic to van der Waals force or hydrogen bond.

Proteins from Rhinella jimi parotoid gland secretion: A comprehensive analytical approach

Toad skin secretions are sources of complex mixtures of bioactive compounds, such as proteins and peptides. Rhinella jimi species is a common toad in the Brazilian northeast, considered by only a few known studies. The experimental design was applied to optimize the protein extraction method from R. jimi parotoid gland secretions. The optimum condition was using 100 mmol L−1 Tris-HCl buffer pH 7.2 under vortexing for 5 min. The FTIR analysis combined with PCA revealed high-protein purity of the extracts, confirming the success of the proposed extraction method. The total protein concentration by the Bradford method was 102.4 and 66.5 mg g−1 on toad poisons from Teresina and Picos, respectively. The comparative proteomic analysis using HPLC-SEC-DAD and 1D SDS-PAGE revealed significant differences in protein abundance. HMW biomolecules showed greater abundance in toads from Teresina, while LMW protein species were more abundant in toads from Picos. The significant difference in amphibian proteome can be attributed to the edaphoclimatic conditions of their habitat. The cytotoxicity of the protein extract from Teresina was higher on the tumor cell lines 4T1 and CT26.WT. These new findings are fundamental for future studies the on identity and biological activity of biomolecules from this noble sample.

Pressurized water extraction – the fast and efficient method for isolation of bioactive proteins from Viscum album leaves

New strategies for the fast, efficient, and environmentally friendly extraction of proteins are required to isolate desired bioactive compounds from a technological point of view. In this study, utilization of the pressurized water extraction (PWE) at low temperature (40 °C) for isolation of mistletoe proteins was investigated. PWE effectiveness, based on protein fingerprints, were compared with those obtained by conventional extractions using 10 mmol L–1 Tris-HCl buffer pH 8.3, 50 mmol L–1 phosphate buffer pH 7, or deionized water. The extracts were precipitated using acetone, trichloroacetic acid (TCA), and 20% (w/v) TCA/acetone and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. PWE was more or equally efficient for isolation of mistletoe proteins than evaluated conventional extraction methods. The proteomic analysis combining mass spectrometry and database searching confirmed the presence of 35 proteins in PWE extracts precipitated by acetone, which was the most compounds identified from all studied extracts. The PWE high extraction power was revealed for multiple viscotoxin isoforms and specific enzymes indispensable for the synthesis of terpenes.

Enhancing immobilization of Aspergillus oryzae ST11 lipase on polyacrylonitrile nanofibrous membrane by bovine serum albumin and its application for biodiesel production

The partially purified lipase from Aspergillus oryzae ST11 was immobilized on the surface-modified electrospun polyacrylonitrile (PAN) nanofibrous membrane. The effects of time and concentrations of glutaraldehyde and bovine serum albumin (BSA) were studied. The immobilized lipase reached the maximum activity at 180 min with the recovered activity of 44.2% (0.22 U/mg-support) in the absence of BSA. After adding 6 mg/mL BSA and 40 mM glutaraldehyde gave the recovered activity at 86.9% (0.43 U/mg-support). It showed the highest stability at pH 7.5 but the soluble lipase was at pH 7.0. In addition, the immobilized lipase was more stable at pH 4.0 and 9.0. The immobilized lipase preserved 56% activity at 70 °C but the soluble lipase had 34% activity. It conserved 86% activity while the soluble lipase had 18% activity after 13 days of storage at 4 °C. The operating parameters such as biocatalyst concentration, water content, and the molar ratio of methanol and palm oil were optimized for biodiesel production. The highest biodiesel conversion (95%) was obtained with the immobilized lipase 15% (w/w), 40% (w/w) 50 mM Tris-HCl buffer pH 7.0 and methanol/palm oil (3.5:1.0). This immobilized lipase could be reused with 82.9% conversion after 10 cycles of batch production.

Adaptation of two-dimensional electrophoresis for muscle tissue analysis

It is important to understand the molecular mechanisms that take place in muscle tissues and to predict meat quality characteristics. One of the most popular methods is two-dimensional electrophoresis, which allows us to visualize, share and identify different molecules, including meat proteins. However, the standard conditions of this method are not universal for all types of raw material, so the authors suggest a new variation of two-dimensional electrophoresis for muscle tissue analysis. Samples were tested by the classical version of isoelectric focusing (cathode buffer in the top and anode buffer in the bottom chamber of the electrophoresis cell) and its variation (anode buffer in the top and cathode buffer in the bottom chamber of the electrophoresis cell). Next, extruded gels were incubated in two different buffer systems: the first was equilibration buffer I (6 M urea, 20% w/v glycerol, 2% w/v SDS and 1% w/v Ditiothreitol in 375 mM Tris-HCl buffer, pH 8.8) followed by equilibration buffer II (6 M urea, 20% w/v glycerol, 2% w/v SDS and 4% w/v iodoacetamide in 375 mM Tris-HCl buffer pH 8.8 and the second, buffer А, consisting of 5 M urea, 2% w/v SDS, 5% v/v mercaptoethanol, 62.5 mM Tris-HCl buffer, pH 6.8 and 0.01% w/v bromophenol blue. Electrophoretic studies of muscle tissue revealed the best protein separation after changing the direction of the current (authors' variation), while no differences were detected after changing incubation buffers.

Identification of blood plasma proteins using heparin-coated magnetic chitosan particles

Heparin was immobilized on magnetic chitosan particles to be used as a tool for human plasma protein identification. Chitosan was magnetized by co-precipitation with Fe2+/Fe3+ (MAG-CH). Heparin was functionalized with carbodiimide and N-hydroxysuccinimide and covalently linked to MAG-CH (MAG-CH-hep). X-ray diffraction confirmed the presence of chitosan and Fe3O4 in MAG-CH. This particle exhibited superparamagnetism and size between 100–300 μm. Human plasma diluted with 10 mM phosphate buffer (pH 5.5) or 50 mM Tris-HCl buffer (pH 8.5) was incubated with MAG-CH-hep, and the proteins fixed were eluted with the same buffers containing increasing concentrations of NaCl. The proteins obtained were investigated by SDS-PAGE, LC/MS, and biological activity tests (PT, aPTT, and enzymatic chromogenic assay). Inhibitors of the serpin family, prothrombin, and human albumin were identified in this study. Therefore, MAG-CH-hep can be used to purify these proteins and presents the following advantages: low-cost synthesis, magnetic separation, ion-exchange purification, and reusability.

Exploration on the bioreduction mechanisms of Cr(VI) and Hg(II) by a newly isolated bacterial strain Pseudomonas umsongensis CY-1.

Despite of significant progress in remediation of Cr(VI) or Hg(II) pollution by microorganisms, study on the reduction of both Cr(VI) and Hg(II) by the same microbial strain was not reported so far, which is actually important for bioremediation of contaminated sites with multiple heavy metals. In this study, Pseudomonas umsongensis CY-1 was newly isolated from chromium-contaminated soil and showed remediation potentials for both Cr(VI) and Hg(II) pollution. The highest Cr(VI) (93.9%) and Hg(II) (82.8%) reduction rates were obtained at the initial concentration of 5mg/L. Comparison between removal by resting cells and heat-treated resting cells demonstrated that P. umsongensis CY-1 removed Cr(VI) and Hg(II) from Tris-HCl buffer (pH 7.0) mainly through reduction instead of adsorption. By comparing the Cr(VI) and Hg(II) reduction rates of different cellular fractions, it was found that Cr(VI) and Hg(II) reductions mainly happened in the cytoplasm of P. umsongensis CY-1, which were further demonstrated by Transmission electron microscopy (TEM) analysis. Furthermore, analysis of X-ray photoelectron spectroscopy demonstrated that the reduction products of Cr(VI) and Hg(II) were mainly in the form of Cr(III) and Hg (0), respectively. The findings in this study will provide a guide for further insights in the bioremediation of contaminated sites with multiple heavy metals.

Purification and Properties of an Esterase from Bacillus licheniformis and it’s Application in Synthesis of Octyl Acetate

Background: Esterase plays a major role in the degradation of natural materials, industrial pollutants and also provides an immense contribution to the eco-friendly approaches in various industrial applications. Objective: In the present study, extracellular esterase from bacterial isolate Bacillus licheniformis was purified, characterized and used in the synthesis of octyl acetate. Methods: Purification of esterase from Bacillus licheniformis was achieved using Sephadex G-75 column chromatography. Gas chromatography was used to analyze the octyl acetate synthesis. Results: The enzyme was salted out using ammonium sulphate precipitation and 60-70% saturation gave maximum specific activity of the enzyme during precipitation. A purification fold of 6.46 and yield of 9.69% was achieved when esterase from Bacillus licheniformis was purified using Sephadex G-75 column chromatography. Native as well as SDS-PAGE analysis gave a single band of 42 kDa. This showed that the enzyme was purified to homogeneity and it was a monomer with molecular weight of 42 kDa. Biochemical characterization of the enzyme revealed that it had optimum temperature of 45°C in 0.1 M Tris-HCl buffer of pH 8.0. On optimizing different parameters, such as molar ratio of reactants, incubation time, temperature, and amount of protein, the % yield of octyl acetate was found to be 77.3%. Conclusion: In this work, simple method was used to purify esterase and the enzyme was further used in producing esters/products of commercial value within a reasonably short period of 12 h with a maximum yield of 77.3%.

Improvement of enzymatic performance of Asclepias curassavica L. proteases by immobilization. Application to the synthesis of an antihypertensive peptide

The aim of this work was to study different immobilization strategies on silica supports in order to obtain robust biocatalysts from latex proteases of Asclepias curassavica L., a South American native plant. Immobilized enzyme performance was evaluated under harsh reaction conditions such as the synthesis of the antihypertensive peptide N-α-CBZ-Val-Gly-OH.Proteases from A. curassavica, named asclepain, were immobilized (0.51–5.56 mg of protein/ g of support) in non-functionalized silica (S), in glyoxyl-silica (GS) and in octyl-glyoxyl-silica (OGS), by adsorption, and multi-point covalent attachment on mono and hetero-functional supports, respectively, under previously determined optimal immobilization conditions. Immobilization yields were expressed as activity yield (Ya) and protein yield (Yp).Asclepain-OGS showed the highest Ya (178 ± 1.62 %) meaning an expressed activity 1.8 times higher than the offered activity, while Yp was 75 ± 0.4 %. Ya for asclepain-S and -GS were 64 ± 1.45 % and 16 ± 0.37 %, respectively. Best results were attributed to the ability of OGS support to guide the enzyme before covalent attachment, increasing its reactivity. Asclepain-OGS led to product yield of 95.5 ± 0.14 %, five times higher than soluble asclepain in the synthesis of N-α-CBZ-Val-Gly-OH, after 3 h in 30 % methanol in 0.1 M Tris-HCl buffer pH 8.

Enzyme adsorption-desorption and evaluation of various cellulase recycling strategies for steam pre-treated Eucalyptus enzymatic degradation

One of the biggest challenges hindering the successful commercialisation of biofuels is the high dosages of enzymes still required to hydrolyse lignocellulose into fermentable sugars. Enzyme recycling has been recognised as a promising approach to alleviate this problem by reducing enzyme costs and improving the profitability of the bioconversion process. However, developing an effective recycling system is a challenging process and requires an in-depth understanding of enzyme-substrate interactions. In this context, this study investigated the adsorption, desorption and re-adsorption of a cellulase mixture (CelMix) to various lignocellulosic components to gain insights into enzyme-substrate interactions, in order to develop an effective enzyme recycling strategy. Lignin-rich residues exhibited a higher adsorption capacity for CelMix (~ 38% adsorption) compared to Avicel cellulose (15.6% adsorption). The recovery of CelMix from lignin, steam pre-treated Eucalyptus and cellulose by various chemicals and alkaline washing was examined. Alkaline washing with Tris-HCl buffer (pH 9.0; 0.05 M) was the most effective method for promoting enzyme desorption (90.7% activity) and retained a substantial amount of hydrolytic activity after elution. However, minor activity loss was observed due to irreversible binding, which was further confirmed by SDS-PAGE analysis. Using this information, the feasibility of recovering the enzymes from the solid fraction after enzymatic hydrolysis of steam pre-treated Eucalyptus was evaluated by two different approaches: (i) re-adsorption of the entire hydrolysed insoluble biomass fraction (no desorption) to fresh biomass (recycling approach 1-RA1) and (ii) re-adsorption of alkaline elution desorbed enzymes from hydrolysed biomass to fresh biomass (recycling approach 2—RA2). The recycling performance of RA1 and RA2 reached > 95% of the initial sugar liberation for three continuous rounds, whilst successful reduction in enzyme loading by 50% and 40% for RA1 and RA2, respectively, was achieved. Therefore, this study shows that enzyme recycling presents a simple and effective pathway for increasing enzyme productivity, improving the economic feasibility of fermentable sugar production for biofuel.

Corrigendum

Corrigendum

An easy and simple separation method for Fc and Fab fragments from chicken immunoglobulin Y (IgY)

Antigen-binding (Fab) and crystallizable (Fc) fragments are the active components of yolk immunoglobulin (IgY), which have been widely used in the pharmaceutical field. However, the common purification methods for the Fab and Fc fragments use combinations of multi-columns are complex and time-consuming. The objective of this study was to improve the separation efficiency of the Fab and Fc fragments from the hydrolyzed IgY and increase the purity of the isolated Fab and Fc fragments. Natural IgY was hydrolyzed using papain for 6 hr and then treated with 45% saturated ammonium sulfate to remove small molecular-weight-peptides. The fraction containing Fab and Fc fragments was loaded on a DEAE-Sepharose ion exchange column and the Fab fraction was washed out first with 10 mM Tris-HCl buffer (pH 7.6). Then, the Fc fraction bound to the DEAE Sepharose was eluted with 10 mM Tris-HCl buffer (pH 7.6) containing 0.21 M NaCl. The purity of the two fragments was 88.7% and 90.1%, respectively. The results of Western blotting and MS analyses indicated that this method purified Fab and Fc fractions with high purity. This method is easy and simple compared with other methods, and the active fragments separated can be easily used.

Interactions of nitrogen-donor biomolecules with copper(II) complexes in Tris buffer

Comprehensive mole-ratio, kinetic, EPR and X-ray studies were performed to investigate substitution reactions between square-planar [CuCl2(en)] (en = ethylenediamine) and square-pyramidal [CuCl2(terpy)] (terpy = 2,2’:6’,2”-terpyridine) complexes with bio-relevant nucleophiles at 295 K in 0.010 M Tris-HCl buffer (pH = 7.4) in the presence of 0.010 M NaCl. According to mole-ratio and EPR studies, Tris buffer reacts with [CuCl2(en)], and likely the complex geometry remains square-planar. Kinetic results have shown that the order of reactivity of nucleophiles towards [CuCl2(en)] in solution for both reaction steps is 1,2,4-triazole > imidazole > pyrazine. The order of reactivity of the azole and diazine ligands toward [CuCl2(terpy)] for the first reaction step is imidazole > 1,2,4-triazole > pyrazine, while for the second one is pyrazine > 1,2,4-triazole > imidazole. X-ray studies indicate great affinity of ethylenediamine toward Cu(II), which results in crystal structures of [Cu(en)2]Cl2·H2O and [Cu(en)2][ZnCl4], instead of dinuclear or heteronuclear complexes with a bridging pyrazine ligand. The recorded EPR spectra demonstrate that all ligands interact with Cu(II) complexes, except in the case of the [CuCl2(en)] complex, where the competition between ligand and Tris molecules might be observed.