Concentrations Of Guanidine Research Articles

Quantification of guanidine in environmental samples using benzoin derivatization and LC-MS analysis

The recent discovery of guanidine-dependent riboswitches in many microbes raised interest in the biological function and metabolism of this nitrogen-rich compound. However, very little is known about the concentrations of guanidine in the environment. Several methods have been published for quantifying guanidine and guanidino compounds in human urine and blood, often relying on derivatization followed by fluorescence detection. We adapted this analytical approach using benzoin as the derivatization agent to sensitively and selectively quantify guanidine in environmental samples, thereby facilitating future research on the biological and environmental roles of guanidine. This adapted method was applied to human urine, raw wastewater, and biological growth media as relevant matrices. Our liquid chromatography-tandem mass spectrometry analyses of the derivatized solutions identified a different major derivatization product than previously reported. This product was consistently observed across various substrates (guanidine, methylguanidine, and arginine) and derivatization agents (benzoin and anisoin). We observed a constant background signal, restricting our analyses to a lower limit of quantification of 50 nM. Despite this limitation, our method allowed for the quantification of guanidine concentrations significantly lower than those reported in previous derivatization-based studies.•Selective and sensitive detection of guanidine by LC-MS.•Method development and validation for robust detection of guanidine in environmental samples.•Reduction of sample preparation steps and reduced usage of toxic chemicals compared to previous methods.

Postpolymerization Modification by Nucleophilic Addition to Styrenic Carbodiimides.

Carbodiimides are electrophilic functional groups that react with select nucleophiles under mild conditions. However, their potential as platforms for postpolymerization modification has been relatively underexplored. We describe the synthesis and radical polymerization of a styrenic carbodiimide which undergoes rapid nucleophilic addition with primary and secondary alkyl amines under ambient conditions, even in the presence of other protic nucleophiles. The monomer is amenable to both free and controlled radical (co)polymerization, and we further demonstrate the utility of this approach by preparing covalent adaptable networks through guanylation of the styrenic carbodiimide with difunctional amines. These materials exhibit a variation in relaxation times according to both the guanidine structure and concentration, providing a facile means for tuning dynamic behavior.

Development of an optimized colorimetric RT-LAMP for SARS-CoV-2 assay with enhanced procedure controls for remote diagnostics

The coronavirus pandemic accentuated the need for molecular diagnostic tests. A technique highly used to this end is the Polymerase Chain Reaction (PCR)—a sensitive and specific technique commonly used as the gold standard for molecular diagnostics. However, it demands highly trained personnel and high-maintenance equipment and is relatively time-consuming. An alternative is the Loop-Mediated Isothermal Amplification (LAMP) technique, which doesn’t need sample purification or expensive equipment, and is similar to PCR when compared in sensitivity and specificity. In this paper, we developed an optimized colorimetric Reverse Transcriptase Loop-Mediated Isothermal Amplification (RT-LAMP) Point-of-Care test using a portable device to diagnose COVID-19. Variables such as concentration of primers, magnesium sulfate, betaine, hydrochloride guanidine, Bst, and temperature of the reactions were tested. We also created a pipetting quality control system—using a combination of dyes—to avoid false negatives due to a lack of samples added to the reaction test tube. Mineral oil was incorporated in the composition of the RT-LAMP reactions to avoid evaporation when a heating lid isn't available. The final RT-LAMP test is tenfold more sensitive when compared to the WarmStart Colorimetric Master mix from New England Biolabs with a sensitivity of 5 copies per μL.

Correlation of the conformational structure and catalytic activity of the highly thermostable xylanase of Thermomyces lanuginosus PC7S1T

Thermomyces lanuginosus PC7S1T xylanase was purified after four chromatographic steps involving columns of ion exchange and molecular filtration. The purified xylanase revealed the molecular mass to be 21.3 kDa, high specificity for beechwood xylan and its main hydrolysis products were xylobiose and xylotriose. Xylanase exhibited increased activity at pH 6.5 and a temperature of 75 °C and was stable-over a wide range of pHs (100 hours) and temperatures (3 hours at 75 °C). Circular dichroism spectrum deconvolution showed a β-structure-rich secondary structure with a melting temperature of 73 °C. In addition, its secondary structure was preserved, even at extreme pHs and temperatures up to 70 °C in the presence of MnCl2 (1 mM), DTT (5 mM) and high concentrations of guanidine (6 M). On the other hand, changes in the tertiary structure of the enzyme, which was monitored using intrinsic and/or extrinsic fluorescence (ANS probe), in the presence of MnCl2, DTT and extreme values of pH may be related to variations in enzyme activity. Therefore, T. lanuginosus PC7S1T xylanase exhibited resistance to high temperatures, observed through its ability to remain active and demonstrate a high-degree of rigidity and resistance to denaturation; very extreme conditions were necessary in order to change the enzymes’ secondary and/or tertiary structures.

Insights on the participation of Glu256 and Asp204 in the oligomeric structure and cooperative effects of human arginase type I

Arginase (EC 3.5.3.1) catalyzes the hydrolysis of L-arginine to L-ornithine and urea, and requires a bivalent cation, especially Mn2+ for its catalytic activity. It is a component of the urea cycle and regulates the intracellular levels of l-arginine, which makes the arginase a target for treatment of vascular diseases and asthma. Mammalian arginases contain an unusual S-shaped motif located at the intermonomeric interface. Until now, the studies were limited to structural role of the motif. Then, our interest was focused on functional aspects and our hypothesis has been that the motif is essential for maintain the oligomeric state, having Arg308 as a central axis. Previously, we have shown that the R308A mutant is monomeric and re-associates to the trimeric-cooperative state in the presence of low concentrations of guanidine chloride.We have now mutated Asp204 that interacts with Arg308 in the neighbor subunit, and also we mutated Glu256, proposed as important for oligomerization. Concretely, the human arginase I mutants D204A, D204E, E256A, E256Q and E256D were generated and examined. No differences were observed in the kinetic parameters at pH 9.5 or in tryptophan fluorescence. However, the D204A and E256Q variants were monomeric. On the other hand, D204E and E256D proved to be trimeric and kinetically cooperative at pH 7.5, whereas hyperbolic kinetics was exhibited by E256A, also trimeric.The results obtained strongly support the importance of the interaction between Arg255 and Glu256 in the cooperative properties of arginase, and Asp204 would be relevant to maintain the oligomeric state through salt bridges with Arg255 and Arg308.

Selection and analyses of variants of a designed protein suggest importance of hydrophobicity of partially buried sidechains for protein stability at high temperatures.

Computationally designed proteins of high stability provide specimen in addition to natural proteins for the study of sequence-structure stability relationships at the very high end of protein stability spectrum. The melting temperature of E_1r26, a protein we previously designed using the A Backbone-based Amino aCid Usage Survey (ABACUS) sequence design program, is above 110 °C, more than 50 °C higher than that of the natural thioredoxin protein whose backbone (PDB ID 1R26) has been used as the design target. Using an experimental selection approach, we obtained variants of E_1r26 that remain folded but are of reduced stability, including one whose unfolding temperature and denaturing guanidine concentration are similar to those of 1r26. The mutant unfolds with a certain degree of cooperativity. Its structure solved by X-ray crystallography agrees with that of 1r26 by a root mean square deviation of 1.3 Å, adding supports to the accuracy of the ABACUS method. Analyses of intermediate mutants indicate that the substitution of two partially buried hydrophobic residues (isoleucine and leucine) by polar residues (threonine and serine, respectively) are responsible for the dramatic change in the unfolding temperature. It is suggested that the effects of mutations located in rigid secondary structure regions, but not those in loops, may be well predicted through ABACUS mutation energy analysis. The results also suggest that hydrophobic effects involving intermediately buried sidechains can be critically important for protein stability at high temperatures.

Relaxation Properties of Nanocomposites Based on Polyamide 6 and Organoclays

The features of organomodifiedmontmorillonite with different concentrations of guanidine acrylate.The influence of modifiers on the relaxation properties of nanocomposites on the basis of PA6 and organoclays by dynamic mechanical analysis.

Environmental Factors Modulating the Stability and Enzymatic Activity of the Petrotoga mobilis Esterase (PmEst).

Enzymes isolated from thermophilic organisms found in oil reservoirs can find applications in many fields, including the oleochemical, pharmaceutical, bioenergy, and food/dairy industries. In this study, in silico identification and recombinant production of an esterase from the extremophile bacteria Petrotoga mobilis (designated PmEst) were performed. Then biochemical, bioinformatics and structural characterizations were undertaken using a combination of synchrotron radiation circular dichroism (SRCD) and fluorescence spectroscopies to correlate PmEst stability and hydrolytic activity on different substrates. The enzyme presented a high Michaelis-Menten constant (KM 0.16 mM) and optimum activity at ~55°C for p-nitrophenyl butyrate. The secondary structure of PmEst was preserved at acid pH, but not under alkaline conditions. PmEst was unfolded at high concentrations of urea or guanidine through apparently different mechanisms. The esterase activity of PmEst was preserved in the presence of ethanol or propanol and its melting temperature increased ~8°C in the presence of these organic solvents. PmEst is a mesophilic esterase with substrate preference towards short-to medium-length acyl chains. The SRCD data of PmEst is in agreement with the prediction of an α/β protein, which leads us to assume that it displays a typical fold of esterases from this family. The increased enzyme stability in organic solvents may enable novel applications for its use in synthetic biology. Taken together, our results demonstrate features of the PmEst enzyme that indicate it may be suitable for applications in industrial processes, particularly, when the use of polar organic solvents is required.

The influence of polyhexamethylene guanidine derivatives introduced into polyhydroxybutyrate on biofilm formation and the activity of bacterial enzymes

Escherichia coli and Staphylococcus aureus were able to produce biofilm on the surface of polyhydroxybutyrate (PHB), but their abundance depended on type and the concentrations of the polyhexamethylene guanidine (PHMG) derivatives introduced in PHB. Different types of PHMG derivatives inhibited S. aureus ATCC 6538P biofilm formation, but PHB with PHMG salt of sulfanilic acid stimulated E. coli ATCC 8739 biofilm formation. The presence of all PHMG derivatives decreased significantly the number of viable cells of the test bacteria directly proportional to the concentration of the biocidal agent. PHMG derivatives affected the activity of microbiological hydrolases with different degrees. Some of them (PHB with PHMG stearate) stimulated activity of E. coli ATCC 8739 hydrolases, other (PHB with the PHMG salt of sulfanilic acid) inhibited activity of the S. aureus ATCC 6538P hydrolases. The PHMG derivatives introduced in PHB also inhibited the activity of bacterial dehydrogenases.

Modification of AxSYM Human Immunodeficiency Virus Assay to Identify Recent Human Immunodeficiency Virus Infections in Korean Human Immunodeficiency Virus-Positive Individuals

ObjectivesTo estimate human immunodeficiency virus (HIV) incidence using HIV avidity assays in Korea, we established a serological testing method to differentiate recent HIV infections from long-standing ones. MethodsWe adopted two incidence assays, the BED HIV-1 incidence test (Calypte Biomedical) and an HIV avidity assay (using Abbott AxSYM HIV Antigen/Antibody Combo), and performed them on Korean HIV samples obtained from 81 HIV seroconverters (n = 193), 135 HIV-positive samples, and three HIV commercial incidence panels (PRB965, PRB933, and PRB601 from SeaCare). To determine the most optimal concentration of the chaotropic agent (Guanidine) and the cutoff value for the avidity assay, we evaluated the sensitivity and specificity of the assay at different concentration levels. ResultsWe determined that the concentration of Guanidine to be used in the avidity assay was 1.5M. The cutoff value of the avidity index (AI) was 0.8, and the sensitivity and specificity were 90.2% and 83.8%, respectively, under this condition. The gray zone for the avidity assay was 0.75–0.85 AI. The mean of coefficient of variation was low, at 5.43%. ConclusionAn optimized avidity assay for the diagnosis of recent HIV infections using Korean samples was established. This assay will be applied to investigate the level of recent infection and will provide basic data to the HIV prevention policy in Korea.

Optoelectronic Data Measuring System for Monitoring Polyhexamethylene Guanidine-Based Decontaminants

A novel method and equipment for measuring concentration of polyhexamethylene guanidine in multicomponent medical disinfectants on the basis of UV spectrometry is described. Basic characteristics and the structural diagram of the automated spectrophotometer are described, and the software is described. The results of the trials of the system in comparison with the results of the conventional photometric method based on the reaction with eosin are given.

THI1, a protein involved in the biosynthesis of thiamin in Arabidopsis thaliana: Structural analysis of THI1(A140V) mutant

In eukaryotes, there are still steps of the vitamin B1 biosynthetic pathway not completely understood. In Arabidopsis thaliana, THI1 protein has been associated with the synthesis of the thiazole ring, a finding supported by the identification of a thiamine pyrophosphate (TPP)-like compound in its structure. Here, we investigated THI1 and its mutant THI1(A140V), responsible for the thiamin auxotrophy in a A. thaliana mutant line, aiming to clarify the impact of this mutation in the stability and activity of THI1. Recently, the THI1 orthologue (THI4) was revealed to be responsible for the donation of the sulfur atom from a cysteine residue to the thiazole ring in the thiamine intermediate. In this context, we carried out a cysteine quantification in THI1 and THI1(A140V) using electron spin resonance (ESR). These data showed that THI1(A140V) contains more sulfur-containing cysteines than THI1, indicating that the function as a sulfur donor is conserved, but the rate of donation reaction is somehow affected. Also, the bound compounds were isolated from both proteins and are present in different amounts in each protein. Unfolding studies presented differences in melting temperatures and also in the concentration of guanidine at which half of the protein unfolds, thus showing that THI1(A140V) has its conformational stability affected by the mutation. Hence, despite keeping its function in the early steps during the synthesis of TPP precursor, our studies have shown a decrease in the THI1(A140V) stability, which might be slowing down the biological activity of the mutant, and thus contributing to thiamin auxotrophy.

On the Mechano-Chemiosmotic Mechanism of Action of Guanidinies on Functional Activity of Mitochondria and Aging

Functional activity of mitochondria in the cell is fully regulated by a complex mixture of dissolved in water substances of the cytosol compound. The concentrations of these substances are different, which is important in processes such as osmoregulation and signaling both of a cell and of mitochondria for ATP synthesis. We studied the action of guanidine on the functional activity of mitochondria. Mitochondria showed a trend to inhibition of oxidative phosphorylation in the presence of different guanidine concentrations. Also, an oscillatory volume changes shrinkage-swelling of mitochondria ¬- are largely abolished and pH changes of incubation medium in addition of ADP are inhibited in the presence of guanidine ion. The mechanism of inhibition of these processes is explained by mechano-chemiosmotic model. In the article, it is discussed that a disruption of the movement of calcium ions (which are moved cyclically between the external environment (cytosol), the matrix and intermembrane space) by various agents, including guanidine leads to disruption of ATP synthesis. We conclude that the L-arginine (2-Amino-5-guanidinopentanoic acid) deficiency in a body leads to a decrease in energy production and, also, it is the cause of aging, type 2 diabetes, cancer and other diseases.

Selection and characterisation of guanidine-resistant mutants of human enterovirus 71

The replication of human enterovirus 71 (HEV71) in cell culture is inhibited by concentrations of guanidine that do not have an observable adverse effect on host cell metabolism. Although the HEV71 non-structural protein 2C is known to play an important role in viral RNA replication, its precise biochemical activities and structure have not been fully determined. Here we describe amino acid substitutions in HEV71 protein 2C that confer resistance to guanidine. Three guanidine-resistant virus populations were independently isolated and found to contain five mutations in protein 2C, one of which, A4657T (2C-M193L), was present in two of the independently selected populations. This mutation was introduced into a HEV71 infectious cDNA clone and was sufficient to confer complete resistance to growth inhibition in the presence of 4mM guanidine. In the first guanidine-resistant population selected, the 2C-M193L mutation occurred in association with an additional mutation, A4459G (2C-I127V), located in the putative cis-acting replication element (cre) of coding region 2C. This mutation conferred only partial guanidine resistance when introduced into the HEV71-26M infectious clone. When the 2C-I127V and 2C-M193L mutations were introduced into HEV71-26M together, the 2C-I127V mutation did not increase the level of guanidine resistance due to the 2C-M193L mutation alone. This study confirms that guanidine resistance can be readily selected in HEV71 and is attributable to mutations within protein 2C.

Guanidine affects differentially the twitch response of diaphragm, extensor digitorum longus and soleus nerve-muscle preparations of mice.

Guanidine has been used with some success to treat myasthenia gravis and myasthenic syndrome because it increases acetylcholine release at nerve terminals through K+, Na+ and Ca2+ channels-involving mechanisms. Currently, guanidine derivatives have been proposed for treatment of several diseases. Studies aimed at providing new insights to the drug are relevant. Experimentally, guanidine (10 mM) induces on mouse phrenic nerve-diaphragm (PND) preparations neurotransmission facilitation followed by blockade and a greatest secondary facilitation after its removal from bath. Herein, we hypothesized that this peculiar triphasic response may differ in muscles with distinct twitch/metabolic characteristics. Morphological alterations and contractile response of PND, extensor digitorum longus (EDL) and soleus (SOL) preparations incubated with guanidine (10 mM) for 15, 30, 60 min were analyzed. Guanidine concentrations of 5 mM (for PND and EDL) and 1 mM (for EDL) were also tested. Guanidine triphasic effect was only observed on PND regardless the concentration. The morphological alterations in muscle tissue varied along time but did not impede the PND post-wash facilitation. Higher doses (20–25 mM) did not increase EDL or SOL neurotransmission. The data suggest a complex mechanism likely dependent on the metabolic/contractile muscle phenotype; muscle fiber types and density/type of ion channels, sarcoplasmic reticulum and mitochondria organization may have profound impact on the levels and isoform expression pattern of Ca2+ regulatory membrane proteins so reflecting regulation of calcium handling and contractile response in different types of muscle.

Improved Protein Hydrogen/Deuterium Exchange Mass Spectrometry Platform with Fully Automated Data Processing

Protein hydrogen/deuterium exchange (HDX) followed by protease digestion and mass spectrometric (MS) analysis is accepted as a standard method for studying protein conformation and conformational dynamics. In this article, an improved HDX MS platform with fully automated data processing is described. The platform significantly reduces systematic and random errors in the measurement by introducing two types of corrections in HDX data analysis. First, a mixture of short peptides with fast HDX rates is introduced as internal standards to adjust the variations in the extent of back exchange from run to run. Second, a designed unique peptide (PPPI) with slow intrinsic HDX rate is employed as another internal standard to reflect the possible differences in protein intrinsic HDX rates when protein conformations at different solution conditions are compared. HDX data processing is achieved with a comprehensive HDX model to simulate the deuterium labeling and back exchange process. The HDX model is implemented into the in-house developed software MassAnalyzer and enables fully unattended analysis of the entire protein HDX MS data set starting from ion detection and peptide identification to final processed HDX output, typically within 1 day. The final output of the automated data processing is a set (or the average) of the most possible protection factors for each backbone amide hydrogen. The utility of the HDX MS platform is demonstrated by exploring the conformational transition of a monoclonal antibody by increasing concentrations of guanidine.

Kinetics of hydrogen evolution reaction on iron and its diffusion through membrane from ethylene glycol solutions

The article discusses the effect of guanidine concentration on the rate of hydrogen evolution reaction (HER) on iron in ethylene glycol-water (0.1 and 50 wt. % H2O, respectively) solutions of HCl with constant ionic strength. In an ethylene glycol environment, guanidine does not vary the rate of HER and the essence of the delayed stage, but rather increases the hydrogen diffusion rate through steel membrane. In the environment with binary solvent, containing 50 wt. % C2H4(OH)2 and electrolyte of the same composition, the discharge rate of (NH2)2CNH2+ ions determines the first stage of HER, the second stage occurs by the Tafel reaction. An increase in the initial concentration of guanidine stimulates HER and slightly varies the hydrogen diffusion flow into metal.

Reactivation of a thermostable lipase by solid phase unfolding/refolding: Effect of cysteine residues on refolding efficiency

Lipase from Geobacillus thermocatenulatus (BTL2) was immobilized in two different matrixes. In one derivative, the enzyme was immobilized on agarose activated with cyanogen bromide (CNBr-BTL2) via its most reactive superficial amino group, whereas the other derivative was covalently immobilized on glyoxyl agarose supports (Gx-BTL2). The latter immobilization protocol leads to intense multipoint covalent attachment between the lysine richest region of enzyme and the glyoxyl groups on the support surface. The resulted solid derivatives were unfolded by incubation under high concentrations of guanidine and then resuspended in aqueous media under different experimental conditions. In both CNBr-BTL2 and Gx-BTL2 derivatives, the oxidation of Cys residues during the unfolding/refolding processes led to inefficient folding for the enzyme because only 25–30% of its initial activity was recovered after 3 h in refolding conditions. Dithiothreitol (DTT), a very mild reducing agent, prevented Cys oxidation during the unfolding/refolding process, greatly improving activity recovery in the refolded forms. In parallel, other variables such as pH, buffer composition and the presence of polymers and other additives, had different effects on refolding efficiencies and refolding rates for both derivatives. In the case of solid derivatives of BTL2 immobilized on CNBr-agarose, the surface's chemistry was crucial to guarantee an optimal protein refolding. In this way, uncharged protein vicinities resulted in better refolding efficiencies than those charged ones.

Influence of guanidine on kinetics of hydrogen evolution reaction on iron and its diffusion through steel membrane in acidic chloride media

Connection between kinetics of hydrogen evolution reaction on iron and concentrations of hydrogen ions (at constant ionic strength equal to 1) and guanidine has been studied in the aqueous (hydrogen ions discharge is rate determining stage) and ethylene glycol+water (10wt.% H2O, recombination of adsorbed hydrogen is rate determining stage) acidic chloride solutions. Guanidine (NH2)2CNH is entirely protonated in the studied conditions. But in the aqueous solutions, a rate of its cathode reduction is insignificant and main proton donor is H3O+-ions. In ethylene glycol solutions a rate of guanidine ions discharge is larger, that though does not determine a nature of rate determining stage. Observed regularities are compared with hydrogen diffusion rate iH through the carbon steel membrane. iH value insignificantly depends on guanidine concentration in aqueous media, especially in the range of its small concentrations. In the ethylene glycol media such a dependence is considerable up to 1mM guanidine concentration.. Nature of rate determining stage of hydrogen evolution reaction does not determine a character of iH=f(CHCl) and iH=f(Cguanidine) dependences.

Kinetic Stability May Determine the Interaction Dynamics of the Bifunctional Protein DCoH1, the Dimerization Cofactor of the Transcription Factor HNF-1α,

The two disparate functions of DCoH1 (dimerization cofactor of HNF-1)/PCD (pterin-4a-carbinolamine dehydratase) are associated with a change in oligomeric state. DCoH dimers enhance the activity of the diabetes-associated transcription factor HNF-1α (hepatocyte nuclear factor-1α), while the PCD activity of DCoH1 homotetramers aids in aromatic amino acid metabolism. These complexes compete for the same interface of the DCoH dimer. Formation of the DCoH1/HNF-1α complex requires cofolding. The homotetramer of the DCoH1 paralogue, DCoH2, interacts with HNF-1α through simple mixing. To further investigate regulation of DCoH/HNF-1α complex formation, we measured the stability of the DCoH1 homotetramer through unfolding studies by intrinsic tryptophan fluorescence. DCoH2 unfolding is reversible. Surprisingly, the DCoH1 homotetramer is resistant to guanidine unfolding but refolds at a much lower guanidine concentration. We show that a point mutation at the DCoH1 tetramer interface, Thr 51 Ser, overcomes the dissociation barrier of the homotetramer and increases the interaction with HNF-1α. The 1.8 Ǻ resolution crystal structure of DCoH1 T51S shows the presence of an ordered water molecule at the tetramer interface, as in DCoH2, which may destabilize the homotetramer. The equilibrium unfolding data were fit to a two-state model with no apparent intermediate. Folding intermediates were detectable by size exclusion chromatography. For wild-type DCoH1 the intermediates changed with time, suggesting a kinetic origin for the unfolding barrier of the homotetramer. We propose an unfolding pathway in which the tetramer unfolds slowly, but the dimer folds reversibly. Implications for regulation of DCoH1/HNF-1α complex formation are discussed.