Tryptophan exposure and accessibility in the chitooligosaccharide-specific phloem exudate lectin from pumpkin (Cucurbita maxima). A fluorescence study

The exposure and accessibility of the tryptophan residues in the chitooligosaccharide-specific pumpkin (Cucurbita maxima) phloem exudate lectin (PPL) have been investigated by fluorescence spectroscopy. The emission λ_max of native PPL, seen at 338 nm was red-shifted to 348 nm upon denaturation by 6 M Gdn.HCl in the presence of 10 mM β-mercaptoethanol, indicating near complete exposure of the tryptophan residues to the aqueous medium, whereas a blue-shift to 335 nm was observed in the presence of saturating concentrations of chitotriose, suggesting that ligand binding leads to a decrease in the solvent exposure of the tryptophan residues. The extent of quenching was maximum with the neutral molecule, acrylamide whereas the ionic species, iodide and Cs⁺ led to significantly lower quenching, which could be attributed to the presence of charged amino acid residues in close proximity to some of the tryptophan residues. The Stern–Volmer plot for acrylamide was linear for native PPL and upon ligand binding, but became upward curving upon denaturation, indicating that the quenching occurs via a combination of static and dynamic mechanisms. In time-resolved fluorescence experiments, the decay curves could be best fit to biexponential patterns, for native protein, in the presence of ligand and upon denaturation. In each case both lifetimes systematically decreased with increasing acrylamide concentrations, indicating that quenching occurs predominantly via a dynamic process.     

Novel Amperometric Hydrogen Peroxide Biosensor Based on Horseradish Peroxidase Azide Covalently Immobilized on Ethynyl‐Modified Screen‐Printed Carbon Electrode via Click Chemistry

A novel, simple and versatile protocol for covalent immobilization of horseradish peroxidase (HRP) on screen‐printed carbon electrode (SPCE) based on the combination of diazonium salt electrografting and click chemistry has been successfully developed. The ethynyl‐terminated monolayers are obtained by diazonium salt electrografting, then, in the presence of copper (I) catalyst, the ethynyl modified surfaces reacted efficiently and rapidly with horseradish peroxidase bearing an azide function (azido‐HRP), thus forming a covalent 1,2,3‐triazole linkage by means of click chemistry. All the experimental results suggested that HRP was immobilized onto the electrode surface successfully without denaturation. Furthermore, the immobilized HRP showed a fast electrocatalytic reduction for H₂O₂. A linear range from 5.0 to 50.0 µM in a phosphate buffer (pH 5.5) with detection limit of 0.50 µM and sensitivity of 0.23 nA/µM were obtained. The heterogeneous electron transfer rate constant K_ct was 1.52±0.22 s^?1 and the apparent Michaelis? Menten constant was calculated to be 0.028 mM. The HRP‐functionalized electrode demonstrated a good reproducibility and long‐term stability.     

Regulation of catalytic activity and functionality of hen egg white lysozyme by alkylhydroxybenzenes

The ability of alkylhydroxybenzenes to change the enzyme activity of hen egg white lysozyme and its efficiency in the catalyzed hydrolysis of nonspecific substrates (chitin and yeasts) was demonstrated. A homolog, C₇-AHB, was used as a modifier; this substance increased the lysozyme enzymatic activity over the entire studied range of concentrations. The effect of C₇-AHB concentration (0.125–4.0 mg/ml), species (initial or oxidized), and the time (1–24 h) of lysozyme incubation with C₇-AHB at 25°C in 0.05 M phosphate buffer (pH 7.4) on the lysozyme activity were studied by differential scanning microcalorimetry, as well as the thermodynamic parameters of its denaturation. The kinetic parameters of chitin hydrolysis by the C₇-AHB-modified lysozyme were determined.     

Evaluation of the pH- and thermal stability of the recombinant green fluorescent protein (GFP) in the presence of sodium chloride

The thermal stability of recombinant green fluorescent protein (GFP) in sodium chloride (NaCl) solutions at different concentrations, pH, and temperatures was evaluated by assaying the loss of fluorescence intensity as a measure of denaturation. GFP, extracted from Escherichia coli cells by the three-phase partitioning method and purified through a butyl hydrophobic interaction chromatography (HIC) column, was diluted in water for injection (WFI) (pH 6.0-7.0) and in 10 mM buffer solutions (acetate, pH 5.0; phosphate, pH 7.0; and Tris-EDTA, pH 8.0) with 0.9-30% NaCl or without and incubated at 80-95 degrees C. The extent of protein denaturation was expressed as a percentage of the calculated decimal reduction time (D-value). In acetate buffer (pH 4.84+/-0.12), the mean D-values for 90% reduction in GFP fluorescence ranged from 2.3 to 3.6 min, independent of NaCl concentration and temperature. GFP thermal stability diluted in WFI (pH 5.94+/-0.60) was half that observed in phosphate buffer (pH 6.08+/-0.60); but in both systems, D-values decreased linearly with increasing NaCl concentration, with D-values (at 80 degrees C) ranging from 3.44, min (WFI) to 6.1 min (phosphate buffer), both with 30% NaCl. However, D-values in Tris-EDTA (pH 7.65+/-0.17) were directly dependent on the NaCl concentration and 5-10 times higher than D-values for GFP in WFI at 80 degrees C. GFP pH- and thermal stability can be easily monitored by the convenient measure of fluorescence intensity and potentially be used as an indicator to monitor that processing times and temperatures were attained.     

Folding and self-association of atTic20 in lipid membranes: implications for understanding protein transport across the inner envelope membrane of chloroplasts

Background

We have previously shown that the P. gingivalis HmuY hemophore-like protein binds heme and scavenges heme from host hemoproteins to further deliver it to the cognate heme receptor HmuR. The aim of this study was to characterize structural features of HmuY variants in the presence and absence of heme with respect to roles of tryptophan residues in conformational stability.

Results

HmuY possesses tryptophan residues at positions 51 and 73, which are conserved in HmuY homologs present in a variety of bacteria, and a tryptophan residue at position 161, which has been found only in HmuY identified in P. gingivalis strains. We expressed and purified the wildtype HmuY and its protein variants with single tryptophan residues replaced by alanine or tyrosine residues. All HmuY variants were subjected to thermal denaturation and fluorescence spectroscopy analyses. Replacement of the most buried W161 only moderately affects protein stability. The most profound effect of the lack of a large hydrophobic side chain in respect to thermal stability is observed for W73. Also replacement of the W51 exposed on the surface results in the greatest loss of protein stability and even the large aromatic side chain of a tyrosine residue has little potential to substitute this tryptophan residue. Heme binding leads to different exposure of the tryptophan residue at position 51 to the surface of the protein. Differences in structural stability of HmuY variants suggest the change of the tertiary structure of the protein upon heme binding.

Conclusions

Here we demonstrate differential roles of tryptophan residues in the protein conformational stability. We also propose different conformations of apo- and holoHmuY caused by tertiary changes which allow heme binding to the protein.     

Boron increases the transition temperature and enhances thermal stability of heme proteins

Transition temperature and thermal stability of proteins were studied in the presence and absence of boron. The observed midpoint of thermal denaturation (T _m) of cytochrome c (Cyt c) at pH 9.2 was 68.8 °C, which in the presence of boron increased to 71.0 °C. For metmyoglobin, T _m increased from 79.7 °C in the absence of boron to 83.5 °C in the presence of boron. Boron caused an increase of 10% in the reversibility of thermal denaturation of cytochrome c when compared with control. Activity measurements of the heat treated proteins and T _m suggest an increased thermal stability toward inactivation and denaturation of heme proteins in the presence of boron.     

Amperometric Flow‐Biosensor for Cyanide Based on an Inhibitory Effect upon Bioelectrocatalytic Reduction of Oxygen by Peroxidase‐Modified Carbon‐Felt

A novel, simple and relative highly sensitive amperometric flow biosensor for cyanide was developed by using horseradish peroxidase (HRP)‐adsorbed carbon‐felt (CF), based on an inhibitory effect on the HRP‐catalyzed O₂ reduction. The HRP‐CF showed a sufficient bioelecrocatalytic activity for O₂ reduction in the potential region from 0 to ?0.5 V at pH 5.0, due to a direct electron transfer‐based O₂ reduction process via ferrous‐HRP and compound III. This HRP‐catalyzed O₂ reduction was reversibly inhibited by cyanide, which enabled to fabricate a novel and simple reagentless (i.e., no requirement of the ordinary substrate, H₂O₂, and the electron transfer mediators) flow‐biosensor for cyanide. When air‐saturated 0.1 M phosphate buffer (pH 5.0) was used as a carrier under the applied potential of ?0.2 V vs. Ag/AgCl, the steady‐state base‐current due to the HRP‐catalyzed O₂ reduction was reversibly inhibited by the cyanide injection (200 µL), resulting in peak‐shape current responses. The magnitude of the inhibition peak currents linearly increased with increasing concentrations of cyanide up to 1 µM, and the detection limit was found to be 0.04 µM (S/N=2). The apparent inhibition constant K_i′ was estimated to be 0.87 µM.     

Use of reversible denaturation for adsorptive immobilization of urease

Urease was chosen as a model multimeric protein to investigate the utility of reversible denaturation for immobilization to a hydrophobic support. Of the various procedures investigated, acidic denaturation provided the highest degree of immobilization and enzymatic activity with lowering of K _m (apparent). Exposure of hydrophobic clusters in the protein molecule induced by the acidic pH environment was confirmed by fluorescence studies using 8-anilino-1-naphtalene-sulfonate as a hydrophobic-reporter probe. The catalytic potential of the enzyme at low pH values was dramatically improved with significant heat and pH stability enhancement on immobilization. Furthermore, the immobilized preparation was used successfully in continuous catalytic transformations. Based on the results presented in this article and a recent report involving a relatively more simple monomeric protein, it is suggested that reversible denaturation may be of general utility for immobilization of proteins, which are not normally adsorbed on hydrophobic supports.     

Ultrasensitive chromogen system for Horseradish Peroxidase Using 3-Methyl-2-benzothiazolinone Hydrazone andN-Phenyldiethanolamine

An ultrasensitive chromogenic system for horseradish peroxidase (HRP) is described. The chromogenic substrate system consists of 3-methyl-2-benzothiazolinone hydrazone (MBTH) andN-phenyldiethanolamine (PDEA). The oxidative coupling of MBTH and PDEA using H₂O₂ and HRP, respectively, as the oxidant and catalyst, yields a blue chromophore that most likely is an indamine dye. The chromophore has a rather broad absorption band, with an absorption maximum at 602 nm. In 0.1M phosphate, 0.1M citrate buffer, pH 7.5, the optimal concentrations of MBTH, PDEA, and H₂O₂ are, respectively, 0.5, 25, and 8 mM. Using this assay system, HRP can be determined in lower picomolar levels by either a rate or fixed-time method.     

Thermodynamic study of the thermal denaturation of a globular protein in the presence of different ligands

By means of difference UV-Vis spectra, the thermal denaturation of catalase has been studied in the presence of different surfactants: sodium perfluorooctanoate, sodium octanoate and sodium dodecanoate. These results indicate that hydrogenated surfactants play two opposite roles in the folding and stability of catalase, they act as a structure stabiliser at a low molar concentrations (enhancing T _m) and as a destabilizer at a higher concentrations (diminishing T _m). Meanwhile sodium perfluorooctanoate enhances T _m in the whole concentration range. An approach for the determination of the heat capacity, enthalpy and entropy has been made, finding that for the three studied surfactants, at all concentrations, the enthalpy term dominates the entropy term.     

STEADY-STATE FLUORESCENCE METHOD FOR EVALUATING EXCITED STATE PROTON REACTIONS: APPLICATION TO FLUORESCEIN

Abstract Fluorescein is a complex fluorophore in the sense that it displays four prototropic forms (cation, neutral, monoanion and dianion) in the pH range 1–9. In experiments with fluorescein-labeled proteins we have sometimes observed complex nanosecond emission kinetics, which could be due to conversion of the excited monoanion into the excited dianion through an excited state proton exchange with a proton acceptor in the labeled protein. However, the literature is ambiguous on whether this possible excited state proton reaction of fluorescein does occur in practice. In this article we describe a general steady-state fluorescence method for evaluating excited state proton reactions of simple as well as complex pH-sensitive fluorophores and apply it to evaluate excited state proton reactions of fluorescein. The method depends on finding a buffer that can serve as an excited state proton donor-acceptor but does not significantly perturb ground state proton equilibrium and especially does not form ground (or excited state complexes) with the fluorophore. Our results show that the excited monoanion-dianion proton reaction of fluorescein does occur in the presence of phosphate buffer, which serves as a proton donor-acceptor that does not significantly perturb ground state proton equilibria. The reaction becomes detectable at phosphate buffer concentrations greater than 20 mM and the reaction efficiency increases with increase in phosphate buffer concentrations. The reaction is most clearly demonstrated by adding phosphate buffer to a solution of fluorescein at constant pH 5.9 with preferential excitation of the monoanion. Under these conditions, the excited monoanion converts to the dianion during its lifetime. The conversion is detected experimentally as an increase in dianion and decrease in monoanion fluorescence intensities with increase in phosphate buffer concentration. The absorption spectrum is not significantly perturbed by the increase in phosphate buffer concentration. To quantitate the reaction, we have recorded titration graphs of fluorescence intensity versus pH for fluorescein solutions at low (5 mM) and high buffer (1 M) concentrations with preferential excitation of the monoanion and preferential detection of the dianion emission. We have also developed theoretical expressions that relate fluorescence intensity to pH in terms of the concentration of the four prototrophic forms of fluorescein, extinction coefficients, fluorescence efficiencies and ground and excited state pK_a. The theoretical expressions give very good fits to the experimental data and allow evaluation of fundamental parameters such as pK_a and fluorescence efficiencies. The analysis of the experimental data shows that the excited monoanion-dianion reaction does not significantly occur at 5 mM phosphate buffer concentration. However, at 1 M buffer concentration the reaction is sufficiently fast that it practically achieves equilibrium during the lifetimes of the excited fluorescein monoanion and dianion. The pK_a* of the excited monoanion-dianion proton reaction is around 6.3. The results and methods presented here should be useful in the development and testing of pH-sensitive labeling fluorophores and fluorescent indicators.     

Denaturation and Leaching Study of Horseradish Peroxidase Encapsulated in Sol-Gel Matrices

Sol-gel matrices have been shown to be relatively inert while preserving the spectroscopic properties and biological activity of the encapsulated proteins. Horseradish peroxidase (HRP) is a hemeprotein widely used in the field of biosensors because of its high specificity for hydrogen peroxide. However, partial inactivation of the protein has been reported when incorporated in aged gels. Whether that inactivation comes from the unfolding of some of the encapsulated proteins or from the leaching of the heme non-covalent active site of HRP is evaluated by absorption and fluorescence spectroscopy. This study shows that the single Trytophan (Trp) fluorescence of HRP may be used to distinguish denaturation processes from leaching of the heme group, as well as to estimate the extent of the denaturation.     

Kinetic resolution of both 1-phenylethanol enantiomers produced by hydrolysis of 1-phenylethyl acetate with <Emphasis Type="Italic">Candida antarctica</Emphasis> lipase B in different solvent systems

The enzymatic resolution of (R, S)-1-phenylethanol produced by hydrolysis of (R, S)-1-phenylethyl acetate catalyzed by immobilized Candida antarctica lipase B (CALB) was successfully carried out in different solvent systems. A systematic screening and optimization of the reaction parameters such as enzyme amount, the nature and the content of organic solvent, pressure and temperature in supercritical carbon dioxide (SC-CO₂) and phosphate buffer, with respect to the conversion rate, were performed. CALB exhibits high enantioselectivity in both tert-butanol with 0.025 mol/l phosphate buffer (pH 7.5) and SC-CO₂ with 0.025 mol/l phosphate buffer (pH 7.5) systems. The conversion rate was 41.2% higher in SC-CO₂ with 0.025 mol/l phosphate buffer (pH 7.5) then in tert-butanol with 0.025 mol/l phosphate buffer (pH 7.5) and the reaction time decreased from 8 h to 90 min.     

A surfactant–heme–sulfonyl imidazole system as a nano-artificial enzyme

The heme–imidazole–sodium dodecyl sulfate (SDS) ternary complex has been designed as a peroxidase-like nano-artificial enzyme, in which the imidazole moiety functions like the histidine ligand in the native horseradish peroxidase (HRP) and increases the reactivity and catalytic efficiency of the designed artificial enzyme by promoting the heterolytic cleavage of hydrogen peroxide. In the present study, three different ligands were used as the imidazole-based ligands in the heme–ligand–SDS ternary system: (1) 1-methylsulfonyl-1H-imidazole, (2) 1-(benzensulfonyl)-1H-imidazole, and (3) 1-tosyl-1H-imidazole (TsIm). The three different ligands gave variable reactivity in the system studied, and the enzymatic activation parameters, using spectrophotometric measurements, showed that the TsIm ligand had a higher catalytic efficiency at 26.38 % of the native HRP efficiency. To investigate the increase in catalytic activity, its mechanism was explored based on the original mechanism of HRP and the structure of its first catalytic intermediate (compound I). Based on the mechanism of HRP and the structure of compound I, a suggested mechanism for Tslm is as follows: the TsIm cation radical makes up part of the compound I structure, which is stabilized in the enzymatic process by charge distribution that is induced via phenyl and methyl groups. Suicide inactivation of heme–TsIm–SDS and heme–imidazole–SDS models was also compared to each other. Suicide inactivation was less exhibited in the presence of TsIm than imidazole in this system unless high concentrations of hydrogen peroxide were used.     

Effect of pH on thermal- and chemical-induced denaturation of GFP

Green fluorescent protein (GFP) is an unusually stable autofluorescent protein that is increasingly being exploited for many applications. In this report, we have used fluorescence spectroscopy to study the effect of pH on the denaturation of GFP with sodium dodecyl sulfate (SDS), urea, and heat. Surprisingly, SDS (up to 0.5%) did not have any significant effect on the fluorescence of GFP at pH 7.5 or 8.5 buffers; however, at pH 6.5, the protein lost all fluorescence within 1 min of incubation. Similarly, incubation of GFP with 8 M urea at 50°C resulted in time dependent denaturation of GFP, but only in pH 6.5 buffer. At higher pH values (pH 7.5 and pH 8.5), the GFP was quite stable in 8 M urea at 50°C, showing only a slight decrease in fluorescence. Heat denaturation of GFP was found to be pH dependent as well, with the denaturation being fastest at pH 6.5 as compared to pH 7.5 or pH 8.5. Like the denaturation studies, renaturation of heat-denatured GFP was most efficient at pH 8.5, followed by pH 7.5, and then pH 6.5. These results suggests that GFP undergoes a structural/stability shift between pH 6.5 and pH 7.5, with the GFP structure at pH 6.5 being very sensitive to denaturation by SDS, urea, and heat.     

Biosensing of Aromatic Amines in Reversed Micelles with Self‐Generation of Hydrogen Peroxide at Glucose Oxidase‐Peroxidase Bienzyme Electrodes

The amperometric biosensing of aromatic amines using a composite glucose oxidase (GOD)‐peroxidase (HRP) biosensor in reversed micelles is reported. Rigid composite pellets of graphite and Teflon, in which GOD and HRP were coimmobilized by simple physical inclusion, were employed for the biosensor design. This design allows the in situ generation of the H₂O₂ needed for the enzyme reaction with the aromatic amines, thus preventing the negative effect that the presence of a high H₂O₂ concentration in solution has on HRP activity. The H₂O₂ in situ generation is performed by oxidation of glucose catalyzed by GOD. The effect of the composition of the reversed micelles, i.e., the nature of the organic solvent used as the continuous phase, the nature and concentration of the surfactant used as emulsifying agent, the aqueous 0.05 mol L^?1 phosphate buffer percentage used as the dispersed phase, and the glucose concentration in the aqueous phase, on the biosensor response was evaluated. Reversed micelles formed with ethyl acetate, a 5% of phosphate buffer (pH 7.0) containing 3.0×10^?3 mol L^?1 glucose, and 0.1 mol L^?1 AOT (sodium dioctylsulfosuccinate), were selected as working medium. Well‐defined and reproducible amperometric signals at 0.00 V were obtained for p‐phenylenediamine, 2‐aminophenol, o‐phenylenediamine, m‐phenylenediamine, 1‐naphthylamine, o‐toluidine and aniline. The useful lifetime of one single biosensor was of 60 days. The trend in sensitivity observed for the aromatic amines is discussed considering the effect of their structure on the stabilization of the radicals formed in the enzyme reaction which are electrochemically reduced. The behavior of the composite bienzyme electrode was also evaluated in a FI (flow injection) system using reversed micelles as the carrier. The suitability of the composite bienzyme electrode for the analysis of real samples was demonstrated by determining aniline in spiked carrots.     

THE EFFECT OF MEDIUM POLARITY ON THE HEMATOPORPHYRIN-SENSITIZED PHOTOOXIDATION OF l-TRYPTOPHAN

Abstract The 83 μM hematoporphyrin (HP)-sensitized photooxidation of 0.1 mM tryptophan in aqueous solution buffered at pH 7.4 or in binary mixtures of phosphate buffer and organic solvents of higher (formamide) or lower (N,N-dimethylformamide, methanol, ethanol, tetrahydrofuran) polarity proceeds by a pure singlet oxygen (¹O₂) mechanism as suggested by azide quenching experiments, the rate-enhancing action of deuterated solvents, and the lack of any significant reaction between triplet HP and tryptophan. Both the first-order rate constant of the photoprocess and the photooxidation quantum yield (φ= 0.011 in phosphate buffer at pH 7.4) increase when the medium polarity is increased (e.g. φ= 0.024 in 90% formamide); this results mainly from the greater quantum yield of ¹O₂ generation and the longer lifetime of ¹O₂. The intrinsic reactivity of ¹O₂ with tryptophan is independent of formamide concentration. A moderate decrease in the medium polarity (e.g. in the range 0-30% methanol) enhances the efficiency of tryptophan photooxidation (φ= 0.014 in 30% methanol) as a result of the enhanced quantum yields of triplet HP and ¹O₂ formation. In contrast, the overall photooxidation rate is depressed at high concentrations of low-polarity organic solvents (e.g. φ= 0.0039 in 90% methanol) due to a 5.5-fold drop of the rate constant for the ¹O₂-tryptophan reaction which counteracts the enhancement of the lifetime and quantum yield of triplet HP and ¹O₂. The solvent composition also affects the equilibria between monomeric and multimeric forms of HP. However, under our experimental conditions, the aggregation state of HP appears to exert only a minor influence on the efficiency of tryptophan photooxidation.     

邻苯二甲酸酐化学修饰对辣根过氧化物酶催化活性的影响

通过采用邻苯二甲酸酐(PA)对辣根过氧化物酶(HRP)的蛋白链进行修饰, 研究了PA化学修饰对HRP的稳定性、催化活性、活性中心结构、酶对底物的亲合性和专一性等催化性质的影响. 结果显示: 酸性条件下(pH=3), 4小时后PA-HRP的催化活性比天然HRP提高了7.5%;碱性条件下(pH=10), 4小时后PA-HRP的催化活性比天然HRP提高了27%. PA-HRP的K_m值为8.16 (mmol/L), 小于天然HRP的K_m值12.99 (mmol/L), 而PA-HRP的k_(cat)/K_m值为7.86(10~4(L/ mmol· min)大于天然HRP的k_(cat)/K_m的6.70(10~4(L/ mmol· min). 这些催化活性和动力学数据表明了PA-HRP与天然HRP相比其稳定性、酶对底物的亲和性和专一性得到了提高. 紫外-可见光谱、拉曼光谱数据显示: 修饰剂PA 改变了天然HRP血红素周围的微环境, 对酶蛋白分子的活性中心结构并没有影响. 差示光谱显示PA修饰剂可以提高酶对底物的亲和力.     

Direct Electron Transfer of Horseradish Peroxidase in Gellan Gum–Hydrophilic Ionic Liquid Gel Film

A new composite film of microbial exocellular polysaccharide‐gellan gum (GG) and hydrophilic room temperature ionic liquid 1‐butyl‐3‐methyl‐imidazolium tetrafluoroborate (BMIMBF₄) was firstly used as an immobilization matrix to entrap horseradish peroxidase (HRP), and its properties were studied by UV/vis spectroscopy, cyclic voltammetry and electrochemical impedance spectroscopy. The results showed that BMIMBF₄ could promote the electron transfer between HRP and electrode surface, and the existence of GG could successfully immobilize BMIMBF₄ on the electrode surface with improved stability. HRP–BMIMBF₄–GG/GCE exhibited a pair of well‐defined and quasireversible cyclic voltammetric peaks in 0.1 M pH 7.0 phosphate buffer solutions at 1.8 V/s, which was the characteristic of HRP Fe(III)/Fe(II) redox couples. The formal potentials (E°′) was ?0.368 V (vs. SCE) and the peak‐to‐peak potential separation (ΔE_P) was 0.058 V. The peak currents were five times as large as those of HRP–GG/GCE. The average surface coverage (Γ*) and the apparent Michaelis‐Menten constant (K_m) were 4.5×10^?9 mol/cm² and 0.67 μM, respectively. The electron transfer rate constant was estimated to be 15.8 s^?1. The proposed electrode showed excellent electrocatalytic activity towards hydrogen peroxide (H₂O₂). The linear dynamic range for the detection of H₂O₂ was 0.05–0.5 μM with a correlation coefficient of 0.9945 and the detection limit was estimated at about 0.02 μM (S/N=3). BMIMBF₄–GG composite film was promising to immobilize other redox enzymes or proteins and attain their direct electrochemistry.     

3‐Indoxyl Phosphate as an Electrochemical Substrate for Horseradish Peroxidase

In this work 3‐indoxyl phosphate (3‐IP), an alkaline phosphatase substrate, is demonstrated to be a suitable substrate for horseradish peroxidase (HRP). HRP catalyzes the oxidation of 3‐IP in presence of hydrogen peroxide (H₂O₂) generating the product indigo blue, which is an aromatic heterocycle compound insoluble in aqueous solutions. This product was easily converted into its soluble parent compound indigo carmine (IC) (by addition of fuming sulfuric acid to the reaction media) which has a reversible voltammetric peak at the formal potential of ?0.15 V (vs. Ag pseudo‐reference electrode) when a screen‐printed carbon electrode (SPCE) is used. Parameters that influence the enzymatic reaction, such as pH, temperature, substrate concentration and reaction time have been optimized. Moreover, the enzyme apparent kinetic constants (V_max, K_M) for both substrates (3‐IP and H₂O₂) have been calculated. Indirect measurements of HRP activity in solution were carried out not only by cyclic voltammetry but also using amperometric detection in a flow system. The detection limits were 6.86×10^?12 and 5.68×10^?12 M, respectively. Thus, 3‐IP is the first substrate that could be used for alkaline phosphatase (AP) and HRP, the most common enzymatic labels in affinity assays.