Analysis of the electron transfer pathway in small laccase by EPR and UV-vis spectroscopy coupled with redox titration

Bacterial small laccases(SLAC) are promising industrial biocatalysts due to their ability to oxidize a broad range of substrates with exceptional thermostability and tolerance for alkaline p H. Electron transfer between substrate, copper centers, and O2is one of the key steps in the catalytic turnover of SLAC. However, limited research has been conducted on the electron transfer pathway of SLAC and SLAC-catalyzed reactions, hindering further engineering of SLAC to produce tunable biocatalysts for novel applications. Herein, the combinational use of electron paramagnetic resonance(EPR) and ultraviolet-visible(UV-vis) spectroscopic methods coupled with redox titration were employed to monitor the electron transfer processes and obtain further insights into the electron transfer pathway in SLAC. The reduction potentials for type 1 copper(T1Cu), type 2 copper(T2Cu) and type 3copper(T3Cu) were determined to be 367 ± 2 mV, 378 ± 5 m V and 403 ± 2 mV,respectively. Moreover, the reduction potential of a selected substrate of SLAC, hydroquinone(HQ), was determined to be 288 mV using cyclic voltammetry(CV). In this way, an electron transfer pathway was identified based on the reduction potentials. Specifically,electrons are transferred from HQ to T1Cu, then to T2Cu and T3Cu, and finally to O2.Furthermore, superhyperfine splitting observed via EPR during redox titration indicated a modification in the covalency of T2Cu upon electron uptake, suggesting a conformational alteration in the protein environment surrounding the copper sites, which could potentially influence the reduction potential of the copper sites during catalytic processes. The results presented here not only provide a comprehensive method for analyzing the electron transfer pathway in metalloenzymes through reduction potential measurements, but also offer valuable insights for further engineering and directed evolution studies of SLAC in the aim for biotechnological and industrial applications.

Determination of aniline in environmental water samples by alternating-current oscillopolarographic titration

A new method for the determination of aniline in environmental water based on oscillopolarographic titration was presented in this paper. Several factors including the kind, concentration, and volume of acid, the dosage of potassium bromide, the temperature and concentration of concomitant substances were investigated in detail. The experimental results indicated that this method was simple, rapid, and sensitive. The linear range was 8.367 × 10(?4) to 2.789 × 10(?2) mol L(?1), the relative standard deviation (R.S.D.) was lower than 0.96%, and the spiked recoveries of aniline in environmental water samples were in the range of 99.4–106.9% under the optimal conditions. The results indicated that the present method could be used as an alternative method for aniline determination in realworld water samples.

Study of Complexes of Lanthanum with Amino Acids by Titration Calorimeter

The stability constants and thermodynamic functions for complexes of lanthanum with eight kind of amino acids according to 1∶1 and 1∶2 in proportion have been determined by titration calorimeter at 298.15 K. The enthalpy change makes a predominant contribution to the stability of these complexes. The ring in amino acid associated with lanthanum ion helps to enhance the stability of complexes. Steric effects between rings in complexes leads to that the equilibrium constants of reaction of the complexes (1∶2) is much less than that of the complexes (1∶1).

Determination of pKa values of alendronate sodium in aqueous solution by piecewise linear regression based on acid-base potentiometric titration

As a mono-sodium salt form of alendronic acid,alendronate sodium presents multi-level ionization for the dissociation of its four hydroxyl groups.The dissociation constants of alendronate sodium were determined in this work by studying the piecewise linear relationship between volume of titrant and p H value based on acidbase potentiometric titration reaction.The distribution curves of alendronate sodium were drawn according to the determined p Ka values.There were 4 dissociation constants(pKa_1=2.43,pKa_2=7.55,pKa_3=10.80,pKa_4=11.99,respectively) of alendronate sodium,and 12 existing forms,of which 4 could be ignored,existing in different p H environments.

Grey relational analysis on the relation between marine environmental factors and oxidation-reduction potential

The effects of marine environmental factors-temperature （T）, dissolved oxygen （DO）, salinity （S） and pH--on the oxidation-reduction potential （ORP） of natural seawater were studied in laboratory. The results show an indistinct relationship between these four factors and the ORE but they did impact the ORP. Common mathematical methods were not applicable for describing the relationship. Therefore, a grey relational analysis （GRA） method was developed. The degrees of correlation were calculated according to GILA and the values of T, pH, DO and S were 0.744, 0.710, 0.692 and 0.690, respectively. From these values, the relations of these factors to the ORP could be described and evaluated, and those of T and pH were relatively major. In general, ORP is influenced by the synergic effect of T, DO, pH and S, with no single factor having an outstanding role.

POTENTIOMETRIC TITRATION OF CALCIUM IN SEAWATER

By using calcium ion selective elatrode (Ca ISE) as indicating electrode, saturated calomel electrode (SCE) as reference electrode and EGTA as titrant, calcium in seawater was determined by potentiometric titration in borate buffer solution. This method can reduce observation errors in the determination of the endpoint, and thus provide better analytical precision(<0.08%) than present complexometric titration.

Conductometric Titration for the Prediction of Conductivity in Solutions with Polymeric Membranes

The analytical technique of conductometric titration is used to characterize polymeric materials.This technique allows obtaining the polymer dry weight capacity(DWC) and the extent of reaction and establishing the optimal number of water molecules per sulfonic groups.In this particular case the polymer material under study was Nafion?117.Two different reactions were made: Neutralization and exchange.Both of them allow obtaining the same result.Conductometric titrations could be considered as a good method to study the neutralization and exchange reactions for polymeric materials used in fuel cells or electrolyzers.The implementation of this analytical technique allows reducing operation time of those types of materials.The waiting time between determinations permits to say that the amount of reactant consumed and products obtained were the same after each addition of titrant.

Extraction Mechanism of Rare Earths with Sec-Octylphenoxy Acetic Acid by Two-Phase Titration Technique

The compositions of the extracted complexes of La, Gd, Er and Y with sec octyl phenoxy acetic acid in heptane and the related apparent extraction equilibrium constants K M were determined using two phase titration technique. The stoichiometric compounds for La, Gd, Er and Y should be LaA 3·2.5HA, GdA 3·3HA, ErA 3·3.1HA and YA 3·4.3HA respectively. And their p K M are 3.43, 3.46, 3.08 and 2.58 respectively.

Rapid determination of phenolic compounds in water samples by alternating-current oscillopolarographic titration

A rapid, simple and sensitive method was demonstrated for the determination of phenolic compounds in water samples by alternating-current oscillopolarographic titration. With the presence of sulfuric acid, phenol could be transferred into a nitroso-compound by reacting with NaNO2. The titration end-point was obtained by the formation of a sharp cut in the oscillopolarographic with infinitesimal NaNO2 on double platinum electrodes. The results showed that phenol had an excellent linear relationship over the range of 4.82×10^-6 -9.65×10^-3 mol/L, the RSD of the proposed method was lower than 1.5%, and the spiked recoveries of three real water samples were in the range of 95.6%-106.9%.

Pitfalls and Uncertainties of Using Potentiometric Titration for Estimation of Plant Roots Surface Charge and Acid-Base Properties

Amount and properties of roots surface charge are important for nutrient uptake and balance in plants. Roots surface charge markedly varies at different rizosphere conditions (particularly pH and ionic strength), which can markedly alter during vegetation season. Among recently available measuring methods, surface charge-pH dependence of roots (as well as other biological objects) is most easily evaluated by potentiometric titration. Use of this method is also easy at different ionic strengths. Potentiometric titration also allows for estimation of the distribution of charge generating surface groups. However, many applications of this method seem to be based either on incorrect methodical or theoretical approaches. In this paper we discuss the methodical and theoretical backgrounds of the titration method. Basing on experimental titration curves of roots of barley grown in nutrient solution, we show inconsistency of surface charge results obtained at different measuring conditions. Limitations of theoretical interpretations of the results are outlined also.

A Multiparameter Colloidal Titrations for the Determination of Cationic Polyelectrolytes

In water treatment processes and conditioning drinking water, PEs are widely used;however, their environmental impact is still doubtful, since residual concentrations increase organic matter content and represents a potential health hazard. This paper demonstrates a multiparametric study of two colloidal titration methods: spectrophotometric and zeta potential end point detection. The first one was optimized to guarantee the accuracy of cationic commercial PE quantification. It includes the indicator dose optimization using analytical criteria for competing equilibria, a calibration curve for two ranges of CPE concentration (1 - 5 ppm and 5 - 100 ppm) and the interference study of flocculant and Sn in the CPE quantification. The second method provides a physicochemical validation of the electric surface phenomena occurring during the colloidal titration and the end point detection. As an additional contribution the zeta potential titration was discussed and proposed as an alternative method for quantifying CPE when the sample is metal free.

Interaction of Ca^(2+) and soil humic acid characterized by a joint experimental platform of potentiometric titration, UV–visible spectroscopy, and fluorescence spectroscopy

Rocky desertification has become a major environmental issue in the karst region of southwestern China.Karst rocky desertification was more severe in regions of limestone soil than in adjacent regions of other soils,despite the relatively higher soil organic matter(SOM)content in limestone soil.The underlying mechanism remains ambiguous.We speculated that the geochemical characteristics of limestone soils in the karst region plays an essential role,especially the high calcium content of limestone soil.To test this hypothesis,we collected limestone soil samples from a limestone soil profile in the southwestern China karst region and extracted humic acid(HA)from these limestone soil samples.We investigated the interaction of Ca^(2+)and three HA samples on a joint experimental platform,which consists of an automatic potentiometric titrator,a UV–visible spectrometer,and a Fluorescence spectrometer.HA solutions were titrated by Ca^(2+)and optical spectra of the HA solutions were monitored during the titration experiments.The results indicated that:(1)the interaction of Ca^(2+)and HA is a combined process of adsorption and complexation.Adsorption dominated the overall distribution behavior of Ca^(2+),which could be fit by Langmuir and Freundlich isotherm models.Complexation was distinguished only when the concentration of Ca2+is low;(2)the changes of UV–visible spectroscopy and excitation–emission matrix fluorescencespectroscopy spectra of HA samples when they were binding with Ca^(2+)implied the apparent molecular size and structure of HA became larger and more complex;(3)the combination of Ca^(2+)and HA plays an important role in the SOM preservation of limestone soils but the stability of the Ca–HA association was relatively weak.The present study draws attention to maintaining the relatively higher Ca^(2+)concentration in limestone soils in ecologic restoration attempts in karst regions.

Preparation and Characterization of Cathode Materials La_(0.7)Sr_(0.3-x)Ca_xCo_(0.9)Fe_(0.1)O_(3-δ) by Reverse Titration Co-Precipitation Method for ITSOFC

The precursors of La0.7Sr0.3-xCaxCo0.9Fe0.1O3-δ(LSCCF, x=0.05, 0.10, 0.15, 0.20) as the cathode materials for intermediate temperature solid oxide fuel cell (ITSOFC) were prepared by reverse titration co-precipitation method with metal-nitrates as starting materials and mixed alkali (NaOH and Na2CO3) as a precipitating agent. The formation process of LSCCF from the precursors was monitored by TG-DSC, and the crystal structure and particles morphology of the precursors which were calcined at 600, 800, 1000 ℃ for 3 h were characterized using XRD, SEM technologies. Compared with the solid state reaction of constituent oxides, when the pH value of the precipitating solution was in the range of 9.1～9.5, the LSCCF powders from the precursors caclined at 800 ℃ for 3 h had high purity, homogeneous and single perovskite phase. The electrical conductivity of the LSCCF samples sintered at 1200 ℃ for 3 h, which was measured as a function of temperatures from 100 to 800 ℃ by DC four-probe method in air, decreased with x from 0.05 to 0.20. The value of electrical conductivity was almost equal because of Ca2+, Sr2+ co-dopant resulting in the 'mix effect' while x=0.10 or 0.15. The electrical conductivity of all doped samples was higher than 100 S·cm-1 at intermediate temperatures from 500 to 800 ℃, and there was good compatibility between the LSCCF cathode and Ce0.8Sm0.2O2 electrolyte.

Investigation of Oxidation-Reduction Properties of Growing Mediums for Cell Culture of Mammals under the Effect of Cosmo-Geophysical Factors

In model experiments were studied the effect of cosmo-geophysical factors of environment (hypomagnetic conditions during 2 days ≈ 1 mkT;electromagnetic irradiation (10 min - 2 MHz with amplitude 5 V/m and power 30 mkVt, background 2 - 4 mkVt), γ-quantum (10 min—from the source 137Cs) and its combined effect on the physic-chemical properties (ORP and pH) of growing medium for cell culture of mammals as nutrition medium 199 (PanEco, Russia). It was used a clear solution of medium (solution 1) and with the adding of 10% embryo bull serum—model of bio-medium (solution 2). Hypomagnetic conditions evoked the decreasing of ORP and pH value in both solutions, electromagnetic irradiation in the solution 1 which evoked the decreasing of ORP and the increasing of pH value, and in the solution 2, on the contrary, the increasing of ORP with the unchanging pH value. γ-radiation sharply decreased ORP value and didn’t change pH in solution 1, i.e. the reduction properties increased. There is insignificant increasing of ORP value and the decreasing of pH is noted in the solution 2, that it is characterized with the increasing of oxidative properties of solution. Under the combined effect of hypomagnetic conditions and electromagnetic irradiation, the values of investigating parameters in the solution 1 decreased and in the solution 2 increased. It was observed acute decreasing of ORP value in both solutions under the combined effect of hypomagnetic conditions and γ-radiation, i.e. the reductive properties of the solutions increased sharply. In this the concentration H+ significantly decreased, (p γ-radiation led to the decreasing of ORP and pH values in both solutions. Thus, the studying factors significantly change the oxidation-reduction properties of growing mediums. The investigation of the processes in biological mediums plays the important role in the assessment of environment effect during the flight in inter-planet space.

Ultraviolet absorbance titration for the determination of conditional stability constants of Hg(Ⅱ) and dissolved organic matter

Strong interaction between natural dissolved organic matter (DOM) and Hg(Ⅱ) may influence the trans- port, conversion, toxicity and bio-validity of mercury in the environment. In this paper ultraviolet (UV) absorbance titration was employed for the first time for the determination of the conditional stability constants of Hg(Ⅱ) and (DOM). With increasing Hg(Ⅱ) concentrations, the UV absorbance of fulvic acid, humic acid, and DOM in river increases progressively. By linear and non-linear model fitting, the conditional stability constants (lgK) of Hg(Ⅱ) and DOM were worked out to be 3.54?4.93 and 3.64?4.85, respectively. The results are consistent with those acquired by the typical fluorescence quenching titration method, with the maximum relative error of lgK being 2.6% and the average relative error being 0.2%. The UV absorbance titration method has the advantages of rapid determination, simple performance, and it will probably become a new approach to studying interactions between DOM and trace metallic ions.

Modified Titration Intratympanic Gentamicin Injection for Unilateral Intractable Ménière's Disease

This study looked into the efficacy of a modified titration protocol of intratympanic gentamicin injection（ITG） in the patients with unilateral intractable Ménière＇s disease（MD）. Modified titration protocol of ITG at a low dose（20 mg/m L） was administered to 10 patients with definite unilateral intractable MD. After initial first two fixed ITGs on weekly basis,the patients might or might not be given any more injections,depending on the appearance of unilateral vestibular loss（UVL）. ITG was terminated if the patients satisfied the criteria of UVL. All patients were followed-up for at least two years. The effects of ITG on the vertigo attack,functional level scores and postural balance were evaluated. Of the 10 cases,8 showed the sign of UVL after receiving initial two ITGs and were not given any more intratympanic injections,and the other 2 patients were administered three ITGs. A two-year follow-up revealed that complete and substantial vertigo control was achieved in 9 cases,and limited vertigo control in 1 patient. Hearing level was lowered in 2 patients. The posture stability and functional level scores were improved. Our study showed that the modified titration protocol of ITG at a low dose could effectively control vertigo in patients with unilateral intractable MD.

Determination of Formation Constants of Co￣(2+), Ni￣(2+),Cu￣(2+) and Zn￣(2+) Complexes with Humic and Fulvic Acidsby Potentiometric Titration Method

The formation constants of Co￣(2+), Ni￣(2+), Cu￣(2+) and Zn￣(2+) complexes with humic acid (HA) and fulvicacid (FA) in red soil were determined by the potentiometric titration method. The constants as a functionof composition of the complexation solutions were obtained by two graphical approaches respectively Theformation constants decreased with increasing concentration of metal in the solution. The results provideunambiguous evidence for the heterogeneity of the function groups of humic substances. The formationconstants of FA were much smaller than those of HA, and the formation constants of Cu￣(2+) were muchgreater than those of Co￣(2+) , Ni￣(2+) and Zn￣(2+) . The potentiometric titration method for determining formationconstants are also discussed in the article.

Determination of Dimethylallylamine by Titration in Nonaqueous Solvent

The content of dimethylallylamine was determined using glacial acetic acid as solvent, acetic-formic mixture as an anhydrite, perchloric acid-glacial acetic acid as titrant, and 1% crystal violet in acetic acid as indicator in the presence of methylamine and dimethylamine The influences of inert constituents and water on the titration were investigated, and a complete analytical method was determined. The results showed that the determination error of total amines increased with water increasing, while the effect of water on the determination of dimethylallylamine was little when the amount of water was within 5%, the relative error was generally within 1%, and that the end-point was acutely when about 10% chloroform was added. Compared with gas chromatography, this method is simple, convenient and accurate.

Study and Application of a New On-Line Titration Technique

A new on line titration method and device based on a new technique continuous flow titration is described. By the means of electronically controlled switching of a solenoid valve, the main component of the system, the equivalent point of the titration is easily determined. Several kinds of mixing tools were examined, whereby a self made mixing chamber with minimum volume gave best results and was therefore used in the device. The error of the titration is within 0.2% and the relative standard deviation (RSD) is below 1.2%. The results show no difference compared with a commercial device, meanwhile the new on line titration system is cheaper and fully automated and thus easy to hand and less solvent consumption.