Oxidation of branched chain amino acids by HOCl: Kinetics and mechanism.

The kinetics of the chlorination of leucine, isoleucine, and valine (BCAAs) was studied in excess HOCl by stopped-flow and spectrophotometric methods (25 ◦C, I = 1.0 M NaClO4). The intermediates and products were identified and monitored by 1H NMR spectroscopy. It was established that these reactions are fully analogous and proceed according to distinct mechanisms under alkaline and neutral conditions. At high pH, the formation and subsequent rate determining decomposition of N-monochloroamino acid control the process. The decomposition occurs via competing pH-independent and OH--assisted reaction paths and the sequence of chlorination, dichlorination and decarboxylation steps leads to the formation of N-chloroimines and their carbanionic forms, which are in fast acid - base equilibria. The dechlorination of the carbanions yields nitriles as the main products. The hydration of the N-chloro imines produces chloramine and aldehydes which are involved in further oxidation reactions with HOCl. The formation of chloroform and chloroacetaldehyde was confirmed in each system. At pH 7.0, the N-chloro derivatives of BCAAs form immediately and are converted into the corresponding N,N-dichloro species within a few seconds after mixing the reactants. In this reaction, the reactive form of the oxidant is Cl2O. The first-order decomposition of the dichloroamino acids occurs on stopped-flow timescale (k = 0.5 - 0.7 s-1) and yields N-chloroimines which slowly decompose with a characteristic first-order rate constant on the order of a few times 10-5 s-1. The main products are the corresponding nitriles that account for about 80% and 60% of the original amounts of amino acids under neutral and alkaline (cOH- = 5.00 × 10-2 M) conditions, respectively. Aldehydes, carboxylic acids, chloroform and NCl3 were also identified as by-products. The results unequivocally confirm that harmful chlorinated species may form from amino acids long after the chlorination step in water treatment technologies that deteriorates the quality of the finished water. ENVIRONMENTAL IMPLICATION: In source waters, amino acids account for about 75% of the total dissolved nitrogen. Therefore, it is an essential issue how the reactions of these compounds with hypochlorite ion can be controlled to avoid the formation of toxic compounds. The compounds formed from BCAAs are considered to be harmful both under alkaline and neutral conditions (chloroacetaldehyde, chloroform, nitriles). However, some of the intermediates have extended lifetime in these systems and they may also react with other components of raw water during water treatment processes.

3Rs implementation in veterinary vaccine batch-release testing: Current state-of-the-art and future opportunities. A webinar and workshop report.

Regulatory authorities require veterinary batch-release testing to confirm vaccine potency and safety, but these tests have traditionally relied on large numbers of laboratory animals. Advances in vaccine research and development offer increasing opportunities to replace in vivo testing, and some stakeholders have made significant progress in incorporating 3Rs elements in quality control strategies. A three-part event series entitled "3Rs Implementation in Veterinary Vaccine Batch-Release Testing: Current state-of-the-art and future opportunities" was jointly organized by the Animal-Free Safety Assessment Collaboration, HealthforAnimals, and the International Alliance of Biological Standardization. Two webinars and a workshop aimed to outline the state-of-the-art non-animal approaches for veterinary batch-release testing. The events included information on the state of the deletion of obsolete safety testing and the current initiatives implemented by European, North American, and Asian-Pacific stakeholders on 3Rs implementation and regulatory acceptance. The events contributed to a better understanding of the barriers to 3Rs implementation. Participants highlighted the need for open communication, continued collaboration between stakeholders, and international harmonization of regulatory requirements to help accelerate acceptance. Despite the challenges, the countries represented at this three-part event have shared their commitments to advancing the acceptance of alternative methods.

Kinetic Role of Reactive Intermediates in Controlling the Formation of Chlorine Dioxide in the Hypochlorous Acid-Chlorite Ion Reaction.

An advanced experimental protocol is reported for studying the kinetics and mechanism of the complex redox reaction between chlorite ion and hypochlorous acid under acidic condition. The formation of ClO2 is followed directly by the classical two-component stopped-flow method. In sequential stopped-flow experiments, the target reaction is chemically quenched using NaI solution and the concentration of each reactant and product is monitored as a function of time by utilizing the principles of kinetic discrimination. Thus, in contrast to earlier studies, not only the formation of one of the products but the decay of the reactants was also directly followed. This approach provides a firm basis for postulating a detailed mechanism for the interpretation of the experimental results under a variety of conditions. The intimate details of the reaction are explored by simultaneously fitting 78 kinetic traces, i.e., the concentration vs. time profiles of ClO2-, HOCl, and ClO2, to an 11-step kinetic model. The most important reaction steps were identified, and it was shown that two reactive intermediates have a pivotal role in the mechanism. While chlorate ion predominantly forms via the reaction of Cl2O, chlorine dioxide is exclusively produced in reaction steps involving Cl2O2. This study leads to clear conclusions on how to control the stoichiometry of the reaction and achieve optimum conditions to produce chlorine dioxide and to reduce the formation of the toxic chlorate ion in practical applications.

The chlorination of glycine and α-alanine at excess HOCl: Kinetics and mechanism.

The chlorination of the two simplest amino acids at HOCl excess was studied by stopped-flow, conventional spectrophotometric and time resolved 1H NMR kinetic methods at 25 °C. These reactions show distinct characteristics under neutral and alkaline conditions. At high pH, the common feature of the two systems is that the N-dichloroamino carboxylate ion does not form and the overall process is controlled by the initial decomposition of the N-monochloro derivative. Under such conditions, carbanions form in equilibrium acid - base processes and open alternative reaction paths, resulting in enhanced complexity of the corresponding mechanisms. In the case of α-alanine, the formation of acetonitrile and N-chloro acetamide as main products; acetate ion, acetaldehyde, chloroacetaldehyde, chloroform as byproducts; acetamide and N-chloro ethanimine as intermediates was confirmed. In the case of glycine, the final products are formamide and OCN-. Under neutral conditions, monochloroamino acid forms immediately upon mixing the reactants, and subsequently it is converted into dichloroamino acid by Cl2O in a fast process. In considerably slower further reaction steps, acetonitrile and acetate ion form as final products in the α-alanine system, while the chlorination of glycine proceeds to full mineralization. The detailed mechanisms suggested for these reactions postulate the formation of various imines and N-chloro imines which are involved in decarboxylation, dechlorination, hydration and hydrolytic reaction steps.

Genetic diversity and substructuring of the Hungarian merino sheep breed using microsatellite markers.

The Hungarian Merino sheep breed (Ovis aries) is the most significant animal resource of the Hungarian sheep sector which, unfortunately, has gone through a huge reduction in number during the last decades and became endangered in 2014. A modern molecular genetic survey is now becoming more than necessary in order to characterize the within-breed genetic diversity and structure. For that reason, six Hungarian Merino flocks were genotyped in 16 microsatellite markers. In total, 144 different alleles were found and the mean values of observed and expected heterozygosity were 0.714 and 0.705, respectively, suggesting a noticeable genetic variability of the breed. The genetic differentiation of the Hungarian flocks was generally low, as reflected by the estimated total FST value (0.036), the extended pattern of admixture in Structure analysis, as well as, by the noticeable level of genetic clustering in UPGMA and FCA analyses. However, two out of the six studied flocks tended to be genetically more distant. The outcome of our study could be a starting point for a planned breeding strategy of the Hungarian Merino breed, by keeping the within-flock genetic variability in priority, as well as, by preserving the potential genetic uniqueness with close monitoring of the inbreeding.

Synthesis and Characterization of 1,10-Phenanthroline-mono-N-oxides.

N-oxides of N-heteroaromatic compounds find widespread applications in various fields of chemistry. Although the strictly planar aromatic structure of 1,10-phenanthroline (phen) is expected to induce unique features of the corresponding N-oxides, so far the potential of these compounds has not been explored. In fact, appropriate procedure has not been reported for synthesizing these derivatives of phen. Now, we provide a straightforward method for the synthesis of a series of mono-N-oxides of 1,10-phenanthrolines. The parent compounds were oxidized by a green oxidant, peroxomonosulfate ion in acidic aqueous solution. The products were obtained in high quality and at good to excellent yields. A systematic study reveals a clear-cut correlation between the basicity of the compounds and the electronic effects of the substituents on the aromatic ring. The UV spectra of these compounds were predicted by DFT calculations at the TD-DFT/TPSSh/def2-TZVP level of theory.

Assessment of Human Mycotoxin Exposure in Hungary by Urinary Biomarker Determination and the Uncertainties of the Exposure Calculation: A Case Study.

Urinary biomarkers of mycotoxin exposure were evaluated in the case of healthy people (n = 41) and coeliac patients (n = 19) by using a multi-biomarker LC-MS/MS immunoaffinity based method capable to analyse biomarkers of nine mycotoxins, i.e., fumonisin B1 (FB1), fumonisin B2 (FB2), deoxynivalenol (DON), zearalenone (ZEN), ochratoxin A (OTA), Aflatoxin B1 (AFB1), T-2 toxin, HT-2 toxin and Nivalenol (NIV). Urinary biomarker concentrations were used to calculate the probable daily intake (PDI) of fumonisin B1, deoxynivalenol, zearalenone and ochratoxin A and compared with their tolerable daily intake (TDI). The human urinary excretion rate values reported in the literature and the 24 h excretion rate measured in piglets were used to estimate and compare the PDI values of the four mycotoxins. The highest mean biomarker concentrations were found for DON (2.30 ng/mL for healthy people and 2.68 ng/mL for coeliac patients). Mean OTA concentration was significantly higher (p < 0.001) in healthy people compared to coeliac patients. PDI calculated with piglets excretion data exceeded the TDI values by a much smaller percentage than when they were calculated from human data, especially for FB1. The uncertainties arising from the different calculations can be well perceived on the basis of these data.

Use of Proton Pump Inhibitors in Hungary: Mixed-Method Study to Reveal Scale and Characteristics.

BACKGROUND: Due to their efficacy and tolerability, utilization of proton pump inhibitors (PPI) has significantly increased worldwide. Parallel to the clinical benefits, potential long-term side effects have been observed, which, along with increasing medical expenses and potential drug interactions, justifies the analysis of the trends of utilization. OBJECTIVE: The aim of the present study was to show the level, pattern, and characteristics of PPI use. METHODS: We assessed the nationwide use of proton pump inhibitors in ambulatory care based on aggregated utilization data from the National Health Insurance database. The annual PPI utilization was expressed as the number of packages and as number of DDDs per 1,000 inhabitants and per year. For 2018, we estimated PPI exposure as the number of packages and as the number of DDDs per user per year. The annual reimbursement costs of proton pump inhibitors were also calculated. Moreover, three patient-level surveys were carried out in non-gastroenterological inpatient hospital departments to reveal characteristics of proton pump inhibitor use, namely dose, duration, and indication. RESULTS: The PPI utilisation increased from 5867.8 thousand to 7124.9 thousand packages and from 41.9 to 50.4 DDD per 1,000 inhabitants and per day between 2014 and 2018. Nationwide data showed that 14% of the adult population was exposed to proton pump inhibitors in 2018, while among hospitalized patients, the prevalence of proton pump inhibitor use was between 44.5% and 54.1%. Pantoprazole was the most frequently used active ingredient, both in the nationwide data and in the patient-level surveys. In the patient-level survey in majority of patients (71.5%-80.0%) proton pump inhibitors were prescribed for prophylaxis. Many inpatients (29.4%-36.9%) used 80 mg pantoprazole per day. The average number of PPI packages per user was 6.5 in 2018 in the nationwide data. The duration of PPI therapy was typically between 1 and 5 years in the patient-level surveys and nearly 20% of the inpatients had been taking proton pump inhibitors for more than 5 years. CONCLUSIONS: Our data suggests that Hungarian patients receive proton pump inhibitors in high doses and for a long time. Use of proton pump inhibitors beyond their recommended indications was also found.

The Chlorination of N-Methyl Amino Acids with Hypochlorous Acid: Kinetics and Mechanisms.

The formation and decomposition kinetics of N-chloro-N-methyl amino acids were studied to predict the fate and impact of these compounds in water treatment technologies and biological systems. These compounds form in fast second-order reactions between N-methyl amino acids and hypochlorous acid. The comparison of the activation parameters for the reactions of N-methyl substituted and nonsubstituted branched-chain amino acids reveals the transition-state features less organized structure and stronger bonds between the reactants in the reactions with the N-methyl derivatives. This is due to a combined positive inductive effect of the N-methyl group and the alkyl side chain as well as to the steric effects of the substituents. N-Methyl-N-chloro amino acids decompose much faster than the nonsubstituted compounds. The reaction rates do not depend on the pH, and the same final product is formed in the entire pH range. N-Chlorosarcosine is an exception, as it decomposes via competing paths, kdobs = kd + kdOH[OH-], yielding different final products. This feature is most likely due to the lack of an alkyl substituent on the α-carbon atom. Under physiological pH, aldehydes and methylamine form in these reactions, which are not particularly toxic.

The decomposition of N-chloro amino acids of essential branched-chain amino acids: Kinetics and mechanism.

The formation of N-chloro-amino acids is of outmost importance in water treatment technologies and also in vivo processes. These compounds are considered as secondary disinfectants and play important role in the defense mechanism against invading pathogens in biological systems. Adversary effects, such as apoptosis or necrosis are also associated with these compounds and the intermediates and final products formed during their decomposition. In the present study, the decomposition kinetics of the N-chloro derivatives of branched chain amino acids (BCAAs) - leucine, isoleucine, valine - were studied. On the basis of spectrophotometric measurements, it was confirmed that the decomposition proceeds via a spontaneous and an OH- assisted path in each case: kobs = k + kOH[OH-]. 1H, 13C NMR and MS experiments were also performed to identify the products and to monitor the progress of the reactions. It was established that the pH independent and the [OH-] dependent paths lead to the formation of the same aldehyde in each system (isovaleraldehyde, 2-methyl-butyraldehyde, and isobutyraldehyde) as a primary product. Under alkaline conditions, a portion of the aldehydes are converted into the corresponding Schiff-bases by the excess amino acid in a reversible process. A common mechanism was proposed for these reactions which postulates the formation of imines and hemiaminals as reactive intermediates.

Occurrence and Risk Assessment of Fumonisin B1 and B2 Mycotoxins in Maize-Based Food Products in Hungary.

Fumonisins are toxic secondary metabolites produced mainly by Fusarium verticillioides and Fusarium proliferatum. Their toxicity was evaluated, and health-based guidance values established on the basis of both Joint FAO/WHO Expert Committee on Food Additives (JECFA) and European Food Safety Authority (EFSA) recommendations. This study presents the results of fumonisin analyses in different maize- and rice-based food products in Hungary and the potential health risk arising from their dietary intake. In total, 326 samples were measured in 2017 and 2018 to determine fumonisins B1 and B2 levels. Three-day dietary record data were collected from 4992 consumers, in 2009. For each food category, the average concentration values were multiplied by the relevant individual consumption data, and the results were compared to the reference values. With respect to the maximum limits, one maize flour, two maize grits, and two samples of other maize-based, snack-like products had total fumonisin content minimally exceeding the EU regulatory limit. The mean daily intake for all maize-product consumers was 0.045-0.120 µg/kg bw/day. The high intake (95 percentile) ranged between 0.182 and 0.396 µg/kg bw/day, well below the 1 µg/kg bw/day tolerable daily intake (TDI) established by EFSA. While the intake calculations resulted in comforting results, maize-based products may indeed be contaminated by fumonisins. Therefore, frequent monitoring of fumonisins' levels and evaluation of their intakes using the best available data are recommended.

The formation of N-chloramines with proteinogenic amino acids.

In this study, the formation of 17 N-chloramines from proteinogenic amino acids and HOCl was studied by direct kinetic method in the pH = 3-13 range. Thus, the uncertainties associated with the indirect methods used in some of the previous studies were eliminated. Each reaction proceeds according to an overall second order kinetics: v = - k [HOCl][R-NH2] and the rate constants are several times 107 M-1s-1. A very slight correlation was found between the lgk and the pKAA of the amino acids. The results make possible to predict the reactivity order of the amino acids toward HOCl under various conditions. A comparison of the parameters of activation indicates that the presence of a bulky substituent on the side chain close to the α-carbon atom decreases the strength of bonding between the reactants and make the structure more diffuse in the transition state. The chlorination of histidine proceeds via two pH dependent paths presumably leading to the formation of N-chloramine and a side chain chlorinated product. The latter compound may be involved in fast subsequent trans-chlorination reactions. The results presented here resolve earlier discrepancies in the literature and are relevant in chlorination water treatment technologies as well as in the interpretation of in vivo processes involving the formation of N-chloro amino acids in a wide pH range.

Determination of chlorine species by capillary electrophoresis - mass spectrometry.

The applicability of CZE with mass spectrometric detection for the determination of four chlorine species, namely chloride and three stable chlorine oxyanions, was studied. The main aspects of the proper selection of BGE and sheath liquid for the CE-MS determinations of anions with high mobility were demonstrated, pointing out the importance of pH and the mobility of the anion in the BGE. The possibility of using uncoated fused silica capillary and common electrolytes for the separation was shown and the advantage of using extra pressure at the inlet capillary end was also presented. The linear range was found to be 1-100 µg/mL for ClO3- and ClO4- , 5-500 µg/mL for ClO2- , and 25-500 µg/mL for Cl- , but the sensitivity can be greatly improved if larger sample volume is injected and electrostacking effect is utilized. The LOD for ClO3- in drinking water was 6 ng/mL, when very large sample volume was injected (10 000 mbar·s was applied).

Size-Dependent H2 Sensing Over Supported Pt Nanoparticles.

Catalyst size affects the overall kinetics and mechanism of almost all heterogeneous chemical reactions. Since the functional sensing materials in resistive chemical sensors are practically the very same nanomaterials as the catalysts in heterogeneous chemistry, a plausible question arises: Is there any effect of the catalyst size on the sensor properties? Our study attempts to give an insight into the problem by analyzing the response and sensitivity of resistive H2 sensors based on WO3 nanowire supported Pt nanoparticles having size of 1.5±0.4 nm, 6.2±0.8 nm, 3.7±0.5 nm and 8.3±1.3 nm. The results show that Pt nanoparticles of larger size are more active in H2 sensing than their smaller counterparts and indicate that the detection mechanism is more complex than just considering the number of surface atoms of the catalyst.

Outstanding Activity and Selectivity of Controlled Size Pt Nanoparticles Over WO3 Nanowires in Ethanol Decomposition Reaction.

Pt nanoparticles with controlled size of 1.5 and 6.5 nm were anchored onto the surface of WO3 nanowires (WO3NW) as well as on MCF-17 silica. In the case of WO3NW and MCF-17 supported nanoparticles, 1.5 nm Pt nanoparticles were more active in ethanol decomposition reaction at 533 K. 6.5 nm Pt/WO3NW catalyst showed ~6 times higher activity compared to MCF-17 supported 6.5 nm Pt nanoparticles. While MCF-17 supported catalysts produced hydrogen, methane, carbon-monoxide and acetaldehyde, the tungsten-oxide supported Pt nanoparticles produced a huge amount of acetone as well as ethene with a high acetaldehyde selectivity besides H2, CH4 and CO. The hydrogen formation was significantly higher when the Pt size was 1.5 nm. The metallic nanoparticles, the acid sites and the oxidized centers of support play important role in the formation of decomposition products of ethanol.

pH controlled byproduct formation in aqueous decomposition of N-chloro-α-alanine.

N-chloro-amino acids are readily formed in chlorination water treatment technologies. These reactions are also important in biological systems where HOCl plays an important role in the defense mechanism against invading pathogens. The intermediates and the products formed are of primary concern because they may have significant biological activities. In order to clarify intimate details and resolve discrepancies in the literature, the decomposition kinetics of N-chloro-α-alanine (MCA) was studied in the neutral - alkaline pH range by UV-vis spectrophotometry and 1H-NMR method. In contrast to earlier reports, the decomposition reaction proceeds via two distinct reaction paths: kobs1 = kOH[OH-] + k, where kOH = (1.38 ± 0.02) × 10-2 M-1s-1 and k = (2.95 ± 0.09) × 10-4 s-1. In slightly alkaline solution, the sole product is acetaldehyde. Under alkaline conditions, the main product is pyruvate ion, however, N-acetyl-α-alanine is also formed in a subsequent reaction sequence. A detailed kinetic model is postulated which involves the rate determining dissociation of MCA into Cl- and ethanimine which produces acetaldehyde in further reaction steps. Via the OH- assisted path, first a carbanion is formed which undergoes dechlorination and produces iminopropionate ion. This species is transformed into pyruvate ion through hydration and deamination steps.

Kinetics and mechanism of the chromium(vi) catalyzed decomposition of hypochlorous acid at elevated temperature and high ionic strength.

An important reaction step in the industrial production of NaClO3 (electrochemical chlorate process) is the thermal decomposition of HOCl/OCl- to yield ClO3- and Cl-. It is widely accepted that this reaction is accelerated by aqueous chromium(vi) species. A detailed kinetic study was conducted under industrially relevant conditions, i.e. at high ionic strength (6.0 M) and elevated temperature (80 °C), to investigate this phenomenon. The decomposition of hypochlorous acid was followed in the presence of Cr(vi) or phosphate (PO43-) or without any additive. In addition to the beneficial pH buffering effect of Cr(vi), the CrO42- form of chromium(vi) was found to slightly catalyze the decomposition of hypochlorous acid. The overall rate of HOCl decomposition can be expressed as -dcHOCl/dt = kdec[HOCl]2[OCl-] + kcat[HOCl]2[CrO42-]. The corresponding rate constants were determined, kdec = 9.4 ± 0.1 M-2 s-1 and kcat = 4.6 ± 0.8 M-2 s-1, and mechanistic interpretation of the catalytic rate law is given. The contribution of the catalytic path to the overall rate of decomposition changes from ca. 30% at pH = 8 to ca. 70% at pH = 6.

Formation of 1,10-Phenanthroline-N,N'-dioxide under Mild Conditions: The Kinetics and Mechanism of the Oxidation of 1,10-Phenanthroline by Peroxomonosulfate Ion (Oxone).

This paper confirms the unexpected formation of 1,10-phenanthroline-N,N'-dioxide (phenO2) when 1,10-phenanthroline (phen) is oxidized by peroxomonosulfate ion (PMS) in a neutral aqueous solution. The kinetics of oxidation of phen by PMS features a complex pH dependence. In 1.00 M H2SO4, 1,10-phenanthroline-mono-N-oxide (phenO) is the sole product of the reaction. The rate of the N-oxidation is highly dependent on pH with a maximum at pH ∼6.7. The formation of phenO occurs via two parallel pathways: the rate constant of the oxidation of phen (k = 3.1 ± 0.1 M(-1) s(-1)) is significantly larger than that of Hphen(+) [k = (4.1 ± 0.3) × 10(-3) M(-1) s(-1)] because the two N atoms are open to oxidative attack in the deprotonated substrate while an internal hydrogen bond hinders the oxidation of the protonated form. With an excess of PMS, four consecutive oxidation steps were found in nearly neutral solutions. In the early stage of the reaction, the stepwise oxidation results in the formation of phenO, which is converted into phenO2 in the second step. The formation of phenO2 was confirmed by (1)H NMR and ESI-MS methods. The results presented here offer the possibility of designing an experimental protocol for preparing phenO2.

Bacterial expression and/or solid phase peptide synthesis of 20-40 amino acid long polypeptides and miniproteins, the case study of Class B GPCR ligands.

By using two different synthetic techniques several polypeptides interacting with Class B type G-protein coupled receptors were prepared. These polypeptides of different lengths (20 ≤ amino acids ≤ 40), structural and aggregation properties, were prepared both by solid phase peptide synthesis (SPPS) and E.coli bacterial expression. Their purity, synthetic yields, by-products and (15)N/(13)Clabelling characteristics were compared as function of i) the applied method, ii) amino acid length and iii) folding propensities. Their tentative yields, costs and "environmental footprints" were analyzed and found as follows. For unlabelled and short polypeptides (n= 20 aa.) the method of choice is the less environmentally friendly however, quick and effective SPPS. If the polypeptide is (un)folded and/or has no aggregation propensity, then SPPS gives relatively good yield (e.g. 14 ± 4%) and a pure product (>97%). For aggregating polypeptides production yields drop for both methods 4 ± 2% (SPPS) and 2 ± 1% (E. coli), respectively. For longer (n≥ 30 aa.) macromolecules (e.g. miniproteins) bacterial expression efficacy gets higher. Moreover biotechnology is "greener", the resulting in raw material is purer (2.8 ± 1.5 mg). All these advantages for at a lower cost: ~4 €/aa. If isotopic labelling is needed for heteronuclear NMR measurements, bacterial expression is the sole option, due to the high cost of (15)N/(13)C labelled Fmoc(Boc)-L-aa-OH starting materials needed for SPPS. In E.coli uniformly double-labelled, pure polypeptides can be obtained for less than 5-700 €/mg, regardless of the length of the polypeptide chain. Thus, chemists are encouraged to use E.coli expression systems when adequate to make not only proteins but polypeptides and miniproteins as well.

Fluorescence of a Histidine-Modified Enhanced Green Fluorescent Protein (EGFP) Effectively Quenched by Copper(II) Ions. Part II. Molecular Determinants.

The histidine-modified EGFP was characterized as a sensing element that preferentially binds nanomolar concentrations of Cu(2+) in a reversible manner (Kd = 15 nM). This research aims to determine the causes of nanomolar-affinity of this mutant by investigating significant structural and energetic alterations of the chromophore in the presence of different copper ion concentrations. In order to reveal the unknown parts of the quenching mechanism we have elaborated a specific approach that combines theoretical and experimental techniques. The theoretical experiment included the modeling of potential distortions of the chromophores and the corresponding changes in energy using quantum mechanical calculations. Differences between the modeled energy profiles of planar and distorted conformations represented the energies of activation for the chromophore distortions. We found that some values of the experimental activation energies, which were derived from fluorescence lifetime decay analysis (ex: 470 nm, em: 507 nm), were consistent with the theoretical ones. Thus, it has been revealed similarity between the theoretical activation energy (50 kJmol(-1)) for 40° phenolate-ring distortion and the experimental activation energy (52.17 kJmol(-1)) required for histidine-modified EGFP saturation with copper. This chromophore conformation was further investigated and it has been found that the large decrease in fluorescence emission is attributed to the significant charge transfer over the molecule which triggers proton transfer thereby neutralizing the cromophore.