Time-resolved fluorescence study of the single tryptophan in thiocyanate and azide derivatives of horseradish peroxidase: Implication for a p H-induced conformational change in the heme cavity

Detailed pH-dependent steady state and picosecond time-resolved tryptophan fluorescence studies on thiocyanate and azide complexes of horseradish peroxidase have been carried out. The fluorescence decay of the single tryptophan in these species was fitted to a discrete three exponential model. Maximum entropy method analysis also gave three distinct regions of lifetime distributions. The fast subnanosecond lifetime component was found to have > 97% amplitude contribution while other two longer lifetime components have small contributions. Small contributions from the nanosecond lifetime components possibly arise from apoprotein impurity or some small amount of disordered heme conformer of the protein. pH dependence of the fast picosecond lifetime components was found to show a systematic behavior which has been interpreted in the light of obligatory conformation change associated with activation of the enzyme at low pH.     

Oxidation of dibenzothiophene catalyzed by heme-containing enzymes encapsulated in sol-gel glass

We have encapsulated several hemoproteins in the sol-gel glass to catalyze the oxidation reaction of dibenzothiophene (model for organic sulfur compounds in coal) with hydrogen peroxide. In addition to cytochrome c and myoglobin, which have previously been encapsulated in sol-gel glasses, two other hemoproteins, horseradish peroxidase and bovine blood hemoglobin, have now been successfully immobilized in sol-gel media with the retention of their spectroscopic properties. All four hemoproteins studied also demonstrate similar catalytic activities toward the oxidation of dibenzothiophene as compared with the results obtained with the proteins in solution. In the case of encapsulated cytochrome c, the more water-soluble S-oxide was obtained with much higher selectivity over the less water-soluble sulfone (S-oxide/sulfone = 7.1) as compared to what was obtained in the aqueous/organic medium (S-oxide/sulfone = 2.3). Because of the advantage of easy separation of the encapsulated proteins from the liquid reaction mixture, it is clear from these studies that the immobilization of active hemoproteins in the solid glass media could serve as more practical biocatalysts.     

Bioanalytical methods applied to endocrine disrupting polychlorinated biphenyls, polychlorinated dibenzo-p-dioxins and polychlorinated dibenzofurans. A review

The usual methods for determining polychlorinated dibenzo-p-dioxins (PCDD), polychlorinated dibenzofurans (PCDF) and polychlorinated biphenyls (PCB) are generally expensive and time consuming. This fact has favored the development of faster and cheaper techniques, based on immunoassays and bioassays. This paper reviews these bioanalytical methods and their analytical importance at the present moment.     

Enantioselective Sulfoxidations Catalyzedby Horseradish Peroxidase, ManganesePeroxidase, and Myeloperoxidase

Summary.  Horseradish peroxidase (HRP), myeloperoxidase (MPO), and manganese peroxidase (MnP) have been shown to catalyze the asymmetric sulfoxidation of thioanisole. When H₂O₂ was added stepwise to MPO, a maximal yield of 78% was obtained at pH 5 (ee 23%), whereas an optimum in the enantiomeric excess (32%, (R)-sulfoxide) was found at pH 6 (60% yield). For MnP a yield of 18% and a high enantiomeric excess of 91% of the (S)-sulfoxide were obtained at pH 5 and a yield of 36% and an ee of 87% at pH 7.0. Optimization of the conversion catalyzed by horseradish peroxidase at pH 7.0 by controlled continuous addition of hydrogen peroxide during turnover and monitoring the presence of native enzyme as well as of intermediates I, II, and III led to the formation of the sulfoxide in high yield (100%) and moderate enantioselectivity (60%, (S)-sulfoxide). Received November 18, 1999. Accepted January 21, 2000     

The use of a radiation inactivation method for characterization of the structure and catalytic properties of enzymes

The use of a radiation inactivation method for studies of the properties of enzymes of different classes is discussed. The possibilities of characterization of enzymes on the basis of the radiation-chemical parameters concerned are described.Translated fromIzvestiya Akademii Nauk. Seriya Khimicheskaya, No, 12, pp. 2828–2836, December, 1996.     

Methylene green-mediated sensor highly sensitive to hydrogen peroxide based on entrapment of horseradish peroxidase in composite membrane of regenerated silk fibroin and poly(vinyl alcohol)

Introduction The detection and quantitative determination of hydrogen peroxide play importantrole in several fields including biochemistry and environmental chemistry.A highlysensitive H202 sensor is useful to fabricate sensor for various substances by combiningit with hydrogen peroxide—producing oxidases.‘Although electrochemical detection ofH202 has been made via its oxidation or reduction at a variety of electrode materials,these electrodes are susceptible to the interference from electr…     

化学发光单孵多层免疫技术检测粪样中轮状病毒

以酶联免疫吸附分析（ＥＬＩＳＡ）夹心测定粪样中轮状病毒（ＲＶ）实验为模型，将传统又孵式免疫操作变成单孵式，即在包被ＲＶ抗体Ａｂ的微孔中同时加入含ＲＶ的粪样上清液和根过氧化物酶（ＨＲＰ）标记抗ＲＶ抗原共同孵育，其结果在载体表面形成多层酶免疫复合物，以ＨＲＰ催化鲁米诺－对碘苯酚－过氧化氢化学体系作为最终信号检测系统，从而建立起化学发光单孵多层免疫技术（ＣＬ－ＳＩＭＩＴ）。其灵敏度是ＥＬＩＳＡ的１６倍，     

Antioxidant enzymes activity in subcellular fraction of freshwater prawnsM. malcolmsonii andM. lamarrei lamarrei

Subcellular distribution of Superoxide dismutase (SOD), catalase (CAT), selenium (SC) dependent glutathione peroxidase, and Se-independent glutathione peroxidase (GSH-Px) activities were detected in different tissues (hepatopancreas, muscle, and gill) of freshwater prawnsMacrobrachium malcolmsonii andMacrobrahium lamarrei lamarrei. CAT and SOD were found almost equally between the mitochondrial and cytosolic fraction. Both Se-dependent and Se-independent GSH-Px activities were mainly found in cytosolic fraction.     

Investigation of voltammetric enzyme-linked immunoassay based on new system of ODA-H_2O_2-HRP

A voltammetric enzyme-linked immunoassay based on a new system of ODA-H2O2-HRP has first been developed and used in the detection of HRP and labelled HRP. By this method, the enzyme-catalyzing reaction of H2O2 oxidizing odianisidine (ODA) couples the electrode-reduction reaction of the oxidizing product of odianisidine, which produces a sensitive polarographic wave at potential of -0.56V (SCE) in Britton-Robinson buffer solution. In using this polarographic wave, a detection limit to HRP is 3.7×10-12g/mL and a linear range 1.0×10-11-2.0×10-9g/mL. And the mechanisms of the coupling reaction and the process of electro-reduction in the ODA-H2O2-HRP voltammetric enzyme-linked immunoassay system have also been carefully studied.