Horseradish Peroxidase H2O2 Research Articles

Injectable Antiswelling and High-Strength Bioactive Hydrogels with a Wet Adhesion and Rapid Gelling Process to Promote Sutureless Wound Closure and Scar-free Repair of Infectious Wounds.

Developing injectable antiswelling and high-strength bioactive hydrogels with wet tissue adhesiveness and a rapid gelling process to meet the requirements for rapid hemostasis, sutureless wound closure, and scar-free repair of infected skin wounds continues to have ongoing challenges. Herein, injectable, antibacterial, and antioxidant hydrogel adhesives based on poly(citric acid-co-polyethylene glycol)-g-dopamine and amino-terminated Pluronic F127 (APF) micelles loaded with astragaloside IV (AS) are prepared. The H2O2/horseradish peroxidase (HRP) system is used to cause cross-linking of the hydrogel network through oxidative coupling between catechol groups and chemical cross-linking between the catechol group and the amino group. The hydrogels exhibit a rapid gelling process, high mechanical strength, an antiswelling effect, good antioxidant property, H2O2 release behavior, and degradability. In addition, the hydrogels present good wet tissue adhesiveness, high bursting pressure, excellent antibacterial activity, long-term sustained release of AS, and good biocompatibility. The hydrogels perform good hemostasis on mouse liver, rat liver, and rabbit femoral vein bleeding models and achieve much better closure and healing of skin incisions than biomedical glue and surgical sutures. Furthermore, the hydrogel dressing significantly improved the scar-free repair of MRSA-infected full thickness skin defect wounds by modulating inflammation, regulating the ratio of collagen I/III, and improving the vascularization and granulation tissue formation. Thus, AS-loaded hydrogels show huge potential as multifunctional dressings for in vivo hemostasis, sutureless wound closure, and scar-free repair of infected skin wounds.

Synthesis of biphenyl-type and diarylether-type oligostilbenes from the biomimetic oxidation of cyano-resveratrol derivatives

Two trans-cyanostilbene derivatives were prepared, and their oxidative dimerizations promoted by horseradish peroxidase-H2O2 were investigated. An M5–M5-coupled biphenyl-type dimer was first synthesized from the biomimetic oxidative coupling reaction of 8-cyano-resveratrol. A novel M5–MO-4-coupled diarylether-type dimer was obtained through the oxidation of 3,5-dibromo-8-cyano-resveratrol and the following debromination reaction. The cyano group at the C-8 of two cyano-stilbenes had obvious influences on the regioselectivity of their coupling reactions.

Improvement of bromide ions on the degradation of sulfamerazine by horseradish peroxidase-H2O2 system and its interaction mechanisms

Degradation of sulfonamide antibiotics (SAs) by horseradish peroxidase (HRP) is an eco-friendly and sustainable approach but with relatively low efficiency. In this study, we present bromide (Br−) enhances sulfamerazine (SMR) degradation in the process of HRP/H2O2. The removal rate of SMR is positively related to Br− concentration and increases over three times within 4 h as Br− concentration increases from 0 to 1 mM. The mechanism of promoting SMR degradation is mainly through oxidation and substitution by reactive bromine, which comes from the oxidation of bromide ions by HRP/H2O2. Moreover, structure and kinetics analysis of HRP confirm that Br− altered the hydrophobic structure of HRP and made it accessible to bind with SMR. Furthermore, eight degradation products are determined by ESI-Q-TOF-MS mass spectrometry. SMR transformation mainly involves two pathways: one way is hydrolysis and then breaking up of sulfonamide bond to generate small fragments, and another is demethylation and Smile rearrangement firstly, then through hydroxylation, SO2 extrusion, and bromine substitution to form other products. Bacteria growth experiments present the fact that neither the degradation products nor bromate formed in the process inhibited bacteria growth, indicating they have no obvious environmental risk. This study first reports Br− could accelerate the degradation of SMR by HRP and proposes the scheme of bromine species changes and SMR transformation pathways. This study not only develops a novel method to enhance the degradation of sulfonamides, but also provides a reference for the environmental risk assessment of sulfonamides.

Regioselective Biomimetic Synthesis of Dimeric Oxyresveratrol Derivatives

AbstractOxyresveratrol and its methylated derivative as coupling precursors were efficiently prepared in four steps, with Wittig reactions and subsequent isomerization reactions as the key steps. The coupling reactions of oxyresveratrol under various oxidative conditions gave a complex and inseparable mixture of coupling products. The oxidative dimerizations of methylated oxyresveratrols catalyzed by horseradish peroxidase–H2O2 or FeCl3·6H2O in an acetone system predominantly produced the 8–5-coupled and 8–10-coupled dihydrobenzofuran-type dimers, respectively. This regioselective biomimetic strategy might be useful in synthesizing other dimeric oxyresveratrol derivatives.

Oxidative cross-coupling approach to the biomimetic synthesis of the heterodimers of resveratrol and isorhapontigenin

The regioselective oxidative cross-coupling reactions of two different stilbene precursors catalyzed by FeCl3 or horseradish peroxidase-H2O2 in acetone solvent produced three dihydrobenzofuran-type heterodimers. The reductive debrominations of these cross-coupled dimers synthesized two heterodimers of resveratrol and isorhapontigenin, an analogue of (±)-scirpusin A and (±)-gnetuhainin Q.

Multiple effects of sodium dodecyl sulfate on chromogenic catalysis of tetramethylbenzidine with horseradish peroxidase

In this work, the chromogenic catalysis of 3,3’,5,5’-tetramethylbenzidine (TMB) with horseradish peroxidase (HRP) in presence of hydrogen peroxide (H2O2), the most widely used chromogenic reaction in enzyme linked immunosorbent assay (ELISA) was deeply studied in the medium containing sodium dodecyl sulfate (SDS). The effects of SDS on the catalytic efficiency and the kinetic behaviors of chromogenic catalysis of HRP–H2O2–TMB were systematically investigated. It was found that the catalysis of HRP–H2O2–TMB follows a ping-pong kinetic mechanism in SDS solutions based on the theoretical model of double substrates enzymatic kinetics. Particularly, multiple significant effects of SDS in different reaction stages have been summarized as follows: the solubilizer of TMB at the early stage of reaction, the promoter of electron transfer at the happening stage of reaction, the stabilizer of blue chromogens at the end of the reaction as well as the binder of HRP during the course of reaction. The results have shown that the first three effects of SDS prevail when SDS concentration is less than 2.5 mM and leading to the catalytic efficiency increase significantly with SDS concentration. However, once SDS concentration is over 2.5 mM, the bonding of SDS on HRP by the strong electrostatic and hydrophobic interaction render SDS act as inhibitors for HRP. The present work may provide feasible means for purposive tailor the chromogenic catalysis of TMB with HRP and improve its chromogenic performance in biochemical detection.

Cu2+-Modified Boron Nitride Nanosheets-Supported Subnanometer Gold Nanoparticles: An Oxidase-Mimicking Nanoenzyme with Unexpected Oxidation Properties.

In recent years, inorganic biomimetic nanozymes that mimic the activity of natural biological enzymes have attracted extensive research interest, and some mimic enzymes have been successfully applied in the fields of biosensing, catalysis, and oncotherapy. Herein, we report the preparation and mechanism study of a novel nanocomposite, Cu2+-modified hexagonal boron nitride nanosheets-supported subnanometer gold nanoparticles (Au NPs/Cu2+-BNNS). Interestingly, our investigation reveals that Cu2+-BNNS exhibits strong peroxidase mimetic nanoenzyme activity, while Au NPs/Cu2+-BNNS exhibits excellent oxidase-like activity, that is, it can catalyze the oxidation reaction of the substrate in the absence of an oxidant such as H2O2. For example, Au NPs/Cu2+-BNNS can efficiently and selectively oxidize 3,3',5,5'-tetramethylbenzidine (TMB) and 3,3'-dimethylbiphenyl-4,4'-diamine (OT) coloration without the presence of horseradish peroxidase (HRP) and H2O2. It is worthy to note that AuNPs/Cu2+-BNNS-induced TMB coloration only takes 4 min to reach the platform, while the conventional HRP-H2O2 system takes more than 30 min to reach the platform. Further mechanism study shows that the zeta potential, oxidation potential, and steric hindrance of the oxidative chromogenic substrate determine the selectivity of oxidation coloration, while the oxidase-like properties of Au NPs/Cu2+-BNNS are derived from reactive oxygen species generated by the adsorbed oxygen, and Cu2+ ion can synergistically promote the oxidation process. Compared with conventional biological enzymes, Au NPs/Cu2+-BNNS has the advantages of being HRP free and H2O2 free, having high efficiency, low cost, and good stability, and is successfully demonstrated for the detection of carcinoembryonic antigen (a universal cancer biomarker) and H2S (the third gaseous signal molecule).

Self-Crosslinking of Silk Fibroin Using H2O2-Horseradish Peroxidase System and the Characteristics of the Resulting Fibroin Membranes.

Silk fibroin has been widely used in biomedical and clinical fields owing to its good biocompatibility. In the present work, self-crosslinking of fibroin molecules was carried out using the hydrogen peroxide (H2O2)-horseradish peroxidase system, followed by preparation of the fibroin membranes, aiming at improving the mechanical property of fibroin-based material and expanding its applications. P-Hydroxyphenylacetamide (PHAD), as the model compound of tyrosine residues in fibroins, was used to investigate the possibility of horseradish peroxidase (HRP)-catalyzed crosslinking. The results were characterized by means of 1H NMR and UPLC-TQD. The efficacy of enzymatic crosslinking of silk fibroins was examined by determining the changes in the relative viscosity, amino acid compositions, and SEC chromatogram. The obtained data indicated that H2O2-HRP incubation led to PHAD polymerization, and the molecular weight of fibroin proteins was also noticeably increased after the enzymatic treatment. CD and ATR-FTIR spectra revealed that H2O2-HRP treatments had an evident impact on the conformational structure of silk fibroins. The mechanical property and thermal behavior for the modified fibroin membrane were noticeably improved compared to the untreated. Meanwhile, the obtained membrane exhibited good biocompatibility according to the cell growth experiment. The present work provides a novel method for preparation of the fibroin-based materials for biomedical applications.

Multicolor Colormetric Biosensor for the Determination of Glucose based on the Etching of Gold Nanorods.

In this work, 3,3′,5,5′-tetramethylbenzidine(II) (TMB2+), derived from H2O2-horseradish peroxidase (HRP)-3,3′,5,5′-tetramethylbenzidine (H2O2-HRP-TMB) reaction system, was used to etch AuNRs to generate different colors of solution. Many enzyme reactions are involved in the production of H2O2 (e.g., glucose can react with the dissolved oxygen in the presence of glucose oxidase (GOx) to produce H2O2). Given this information, a simple visual biosensor was developed in this study, with glucose as the example target. The detection range of the proposed system varied with the experimental conditions, such as the concentration of GOx and HRP, and enzymatic reaction time. Under the optimized conditions, the longitudinal shift of localized surface plasmon resonances (LSPR) had a linear correlation with the glucose concentration in the range of 0.1~1.0 mM. Meanwhile, the solution displayed a specific color in response to the glucose concentration, thus enabling the visual quantitative detection of glucose at a glance. Compared with the traditional monochromic colorimetry, this multicolor glucose sensor generates various vivid colors, which can be easily distinguished by naked eyes without any sophisticated instrument. Notably, the proposed method has been successfully applied to detect glucose in serum samples with satisfied results.

Two horseradish peroxidase-based modifications result in two milk protein products with ordered secondary structure and enhanced in vitro antigenicity

ABSTRACTIn this study, two horseradish peroxidase (HRP)-based modifications were used to treat whey protein isolate (WPI) and skim milk powder (SKMP) at protein concentration of 50 g/L, pH 7.0, and 37°C, via dityrosine formation in protein molecules. When HRP, glucose oxidase, and glucose were used to treat WPI and SKMP, suitable usages of HRP, glucose oxidase, and glucose, and reaction time were selected from single factor trials based on relative dityrosine contents of the modified products. After that, the selected HRP plus the calculated H2O2 were used to treat WPI and SKMP. Electrophoretic analysis demonstrated that the modified products contained some cross-linked proteins with greater molecular weights. Other analyses showed that the modified products had more ordered secondary structure, and higher in vitro α-lactalbumin and β-lactoglobulin antigenicity than the respective substrates. HRP–glucose oxidase–glucose system was more potent than HRP–H2O2 system to enhance in vitro antigenicity of WPI and SKMP.

A novel strategy to improve the sensitivity of antibiotics determination based on bioelectrocatalysis at molecularly imprinted polymer film electrodes.

A new strategy for the sensitive detection of kanamycin (KA) and other antibiotics based on molecularly imprinted polymer (MIP) and bioelectrocatalysis was developed in the present study. The KA-polypyrrole MIP films were electropolymerized on the surface of pyrolytic graphite (PG) electrodes, with pyrrole (PY) serving as the monomer and KA as the template. Because KA is electro-inactive, electroactive K3[Fe(CN)6] was used as the probe in the cyclic voltammetric (CV) measurements. The difference of the CV reduction peaks of K3[Fe(CN)6] at electrodes between the MIP films after KA removal and KA-rebinding MIP films could thus be used to determine KA quantitatively. When horseradish peroxidase (HRP) and H2O2 were added into the testing solution, the detection sensitivity of the system was greatly amplified because the electrochemical reduction of H2O2 could be catalyzed by HRP and mediated by K3[Fe(CN)6]. With the bioelectrocatalysis amplification, the limit of detection (LOD) for KA fell as low as 28 nM, approximately two orders of magnitude lower than that for the MIP films in the absence of enzymatic catalysis. The strategy demonstrated the generality. Not only KA but also other antibiotics, such as oxytetracycline (OTC), could be determined by this method. More significantly, in addition to the K3[Fe(CN)6]-HRP-H2O2 system, other bioelectrocatalysis systems, such as Fc(COOH)2-GOD-glucose (Fc(COOH)2=ferrocenedicarboxylic acid, GOD=glucose oxidase), could also be used to amplify the CV signal and realize the sensitive detection of KA for the MIP film system, thereby illustrating the great potential and prospects of the strategy.

Chemiluminescence resonance energy transfer imaging on magnetic particles for single-nucleotide polymorphism detection based on ligation chain reaction

A novel ligation chain reaction (LCR) methodology for single-nucleotide polymorphism (SNP) detection was developed based on luminol–H2O2–horseradish peroxidase (HRP)-mimicking DNAzyme–fluorescein chemiluminescence resonance energy transfer (CRET) imaging on magnetic particles. For LCR, four unique target-complement probes (X and X⁎, YG and Y⁎) for the amplification of K-ras (G12C) were designed by modifying G-quadruplex sequence at 3′-end of YG and fluorescein at 5′-end of Y⁎. After the LCR, the resulting products of XYG/X⁎Y⁎ with biotin-labeled X⁎ were captured onto streptavidin-coated magnetic particles (SA-MPs) via specific biotin-SA interaction, which stimulated the CRET reaction from hemin/G-quadruplex-catalyzed luminol–H2O2 CL system to fluorescein. By collecting signals by a cooled low-light CCD, a CRET imaging method was proposed for visual detection and quantitative analysis of SNP. As low as 0.86fM mutant DNA was detected by this assay, and positive mutation detection was achieved with a wild-type to mutant ratio of 10,000:1. This high sensitivity and specificity could be attributed to not only the exponential amplification and excellent discrimination of LCR but also the employment of SA-MPs. SA-MPs ensured the feasibility of the proposed strategy, which also simplified the operations through magnetic separation and separated the reaction and detection procedures to improve sensitivity. The proposed LCR-CRET imaging strategy extends the application of signal amplification techniques to SNP detection, providing a promising platform for effective and high-throughput genetic diagnosis.

A SERS‐active enzymatic product used for the quantification of disease‐related molecules

In this paper, a method for enhancing the analysis of biologic materials was designed. This method incorporated the surface‐enhanced Raman scattering (SERS) technique and an enzyme catalysis‐based bioassay to develop a more efficient analysis procedure. Using this combination, the quick and simple detection of disease‐related molecules (the human cardiac isoform of troponin T, cTnT) in human serum was achieved. This method utilizes enzyme catalysis to develop a H2O2‐horseradish peroxidase (HRP)‐3,3′,5,5′‐tetramethylbenzidine (TMB) chromogenic system, and the final enzymatic product TMB2+ revealed perfect SERS activities. By analyzing a concentration‐dependent SERS spectrum, the quantitative analysis was accomplished. Our findings reveal that the proposed method has a wider linear range and a more sensitive detection limit compared with traditional chromogenic tests. In summary, two existing methods have been combined to create a new model, which has the potential to be used for the bioanalysis and early diagnosis of diseases. Copyright © 2013 John Wiley & Sons, Ltd.

Impacts of Dissolved Organic Matter on the Removal of 17β-Estradiol Using Horseradish Peroxidase

The impacts of dissolved organic matter (DOM) on the removal of 17β-estradiol (E2) in horseradish peroxidase (HRP)-mediated oxidative coupling systems were investigated in this study. The results showed that the removal rate of E2 and the affinity of HRP to E2 had been significantly decreased in the presence of DOM. Compared with urban sludge DOM (USDOM), river sediment DOM (RSDOM), which features relatively low aromaticity and molecular weight and a large number of O-containing groups, more strongly inhibited the E2–HRP–H2O2 reactions than USDOM. The products were analyzed with electrospray ionization mass spectrometry. The results suggested that the self-coupling of E2 was suppressed in the presence of DOM, likely resulting from cross-coupling between DOM and E2 or self-coupling of DOM in the HRP system. The results are useful in understanding the fate of estrogens in natural systems.

In vitro evaluation of the antioxidant activity of liposomal flavonols by the HRP–H2O2–luminol system

Considering that antioxidant flavonols have been reported to be beneficial to human health, but that their low water solubility and bioavailability limit their administration through systemic route, the development of suitable flavonol-carriers is of great importance for clinical therapeutics. The aim of this study was to prepare liposomes containing flavonols or not and evaluate their antioxidant activity. Vesicles were obtained by ethanol injection method and characterized in terms of entrapment efficiency, size and zeta potential. Inhibitory activity of liposomal flavonols on reactive oxygen species generation was assessed in vitro using luminol–H2O2–horseradish peroxidase technique. Antioxidant activity of liposomal flavonols is dependent on concentration and chemical structure of active compound. Quercetin and myricetin are the most active flavonols (IC50 = 0.6–0.9 µmol/L), followed by kaempferol (IC50 = 3.0–4.5 µmol/L) and galangin (IC50 = 4.0–7.0 µmol/L). Our results suggest that antioxidant-loaded liposomes may be promising tools for therapy of diseases where oxidative stress is involved.

Chemiluminometric Determination of the Pesticide Pirimicarb by a Flow Injection Analysis Assembly

A flow injection (FI) method is described for the determination of pirimicarb. It was found that an enhanced chemiluminescence (CL) signal is obtained when employing the luminol–H2O2–horseradish peroxidase (HRP) system. Under the optimum experimental conditions, the enhanced CL intensity was linear with the concentration 4.25–30.75 ng mL−1 (r = 0.997, n = 8) with a relative standard deviation of 0.99%, containing 12.75 ng mL−1 (n = 8). The limit of detection of the investigated compound was 0.12 ng mL−1. The method shows a moderate selectivity against other pesticides (Amitrole, Atrazine, 2,4,5-T, Dichlorprop, and Metamidophos).The proposed method was sensitive, simple, rapid, and successfully applied to the determination of pirimicarb when it is applied in freshwater; the mean recoveries were 98.3–118.5%.

Investigation of voltammetric enzyme-linked immunoassay based on a new system of HAP-H 2O 2-HRP

By introducing heterocyclic compound to immunoassay system as an electrochemical substrate for the fist time, a new voltammetric enzyme-linked immunoassay system of 3-hydroxyl-2-aminopyridine (HAP)-H 2O 2-horseradish peroxidase (HRP) has been developed. HAP was oxidized with H 2O 2 catalyzed by HRP, and the resulting electroactive product produced a sensitive voltammetric peak at potential of −0.36 V (vs . SCE) in Britton–Robinson (BR) buffer solution. The process of the enzyme-catalyzed reaction and the electro-reduction of the product have been investigated in detail. The linear range for detection of free HRP was from 4.0 × 10 −13 to 1.0 × 10 −9 g/mL with a detection limit of 1.2 × 10 −13 g/mL. The new system has been successfully applied for the assay of α-fetoprotein (αFP) in human serum ranging from 0.1 to 200 ng/mL with a detection limit of 0.1 ng/mL, which was 10 times lower than that of traditional spectrophotometric enzyme-linked immunosorbent assay (ELISA) method. HAP-H 2O 2-HRP voltammetric enzyme-linked immunoassay showed a promising alternative approach in the detection of αFP in clinical diagnosis.

Substituent effects of 3,5-disubstituted p-coumaryl alcohols on their oxidation using horseradish peroxidase–H2O2 as the oxidant

The consumption rates of three monolignols (p-coumaryl, coniferyl, and sinapyl alcohols) and eight analogues using horseradish peroxidase (HRP)–H2O2 as an oxidant were measured and compared with the anodic peak potentials thereof measured with cyclic voltammetry. 3-Monosubstituted p-coumaryl alcohols, i.e., 3-methoxy-, 3-ethoxy-, 3-n-propoxy-, and 3-n-butoxy-p-coumaryl alcohols, had faster reaction rates than p-coumaryl alcohol. This is most probably due to the electron-donating effect of alkoxyl groups. However, the reaction rates gradually decreased with an increase in the molecular weight of the alkoxyl groups. Furthermore, t-butoxyl group, which is a very bulky substituent, caused an extreme reduction in the reaction rate, even though its electron-donating effect was almost the same as that of other alkoxyl groups. The reaction rates of 3,5-disubstituted p-coumaryl alcohols, especially 3,5-dimethyl-p-coumaryl alcohol, were very low compared with 3-monosubstituted p-coumaryl alcohols. These results suggest that there are three main factors of hindrance during the approach of monolignols to the active site of HRP. First, from the results of 3-monoalkoxy-p-coumaryl alcohols, it was suggested that the volume of substituents could decrease their oxidation rates. Second, from the results of 3,5-disubstituted p-coumaryl alcohols, it was suggested that local steric hindrance by the amino residues quite near the heme decreased the oxidation rates. Third, from the results of the substrates with hydrophobic substituents at their 3,5-positions, we suggested that hydrophilicity near heme would decrease their oxidation rates.

Inactivation of creatine kinase induced by stilbene derivatives.

Compounds acting as antioxidants to lipids often have a prooxidant effect on DNA or protein. In this study, inactivation of creatine kinase was examined as an indicator of protein damage induced by antioxidative stilbene derivatives, including diethylstilboestrol, resveratrol and tamoxifen, with horseradish peroxidase and hydrogen peroxide (horseradish peroxidase-H2O2). Diethylstilboestrol and resveratrol, but not tamoxifen, rapidly inactivated creatine kinase. Also, creatine kinase in heart homogenate was inactivated by diethylstilboestrol and resveratrol. Tamoxifen, which has no phenolic hydroxyl groups in its structure, was about 10 times less active in protecting lipids and creatine kinase than diethylstilboestrol and resveratrol, suggesting that phenolic hydroxyl groups in diethylstilboestrol and resveratrol of stilbene derivatives are anti- and pro-oxidative. Absorption spectra of these stilbene derivatives rapidly changed during the reaction with horseradish peroxidase-H202. Diethylstilboestrol and resveratrol free radicals emitted electron spin resonance signals and creatine kinase effectively diminished the electron spin resonance signals. These results suggest that free radicals of diethylstilboestrol and resveratrol formed through reaction with horseradish peroxidase-H202 inactivated creatine kinase. Presumably, oxidation of essential cysteine and tryptophan residues lead to inactivation of creatine kinase. Other enzymes, including alcohol dehydrogenase and cholinesterase, were also sharply inhibited by diethylstilboestrol and resveratrol with horseradish peroxidase-H202. Free radicals of diethylstilboestrol and resveratrol seem to mediate between anti- and prooxidative actions.

Selective activity of butyrylcholinesterase in serum by a chemiluminescent assay.

In a previous study, we showed that purified commercial esterase activity can be detected in a chemiluminescent assay based on the hydrolysis of 2-methyl-1-propenylbenzoate (MPB) to 2-methyl-1-propenol, which is subsequently oxidized by the horseradish peroxidase (HRP)-H(2)O(2) system. The purpose of this study was to verify the applicability of this assay to human serum. The existence of an esterase activity capable of hydrolysing MPB is indicated by the fact that the MPB-serum-HRP-H(2)O(2) system consumes oxygen and emits light. Both signals were abolished by prior serum heat inactivation and were preserved when serum was stored at < or =4 degrees C. Addition of aliesterase inhibitors, such as fluoride ion and trichlorfon or the cholinesterase inhibitor eserine, totally prevents light emission. The butyrylcholinesterase-specific substrate benzoylcholine causes a delay in both O(2) uptake and light emission, while the specific acetylcholinesterase substrate, acetyl-beta-methylcholine, had practically no effect. Purified butyrylcholinesterase, but not acetylcholinesterase, triggered light emission. The finding that butyrylcholinesterase is responsible for the hydrolysis of MPB in serum should serve as the basis for the development of a specific chemiluminescent assay for this enzyme.