Electrochemical studies of 1,5-dihydroxynaphthalene as substrate for voltammetric enzyme immunoassay

Immunoassay is one of the biochemical analytical techniques using the specific antigen antibody com-plexation for analytical purposes. It has extensive ap-plication in clinical diagnostics, prevention and cure of diseases, and virus diagnostics. The presentation and progress of immunoassay methodology are one of the greatest achievements of bioanalytical chemistry. It is estimated that several-hundred millions of immuno-analytical determinations are carried out every year all over the world. E…     

Analysis of biomass cellulose in simultaneous saccharification and fermentation processes

A direct method for determining the cellulose content of biomass residues resulting from simultaneous saccharifiaction and fermentation (SSF) experiment has been developed and evaluated. The method improves on classical cellulose assays by incorporating the enzymatic removal of yeast glucans from the biomass residue prior to acid hydrolysis and subsequent quantification of cellulose-derived glucose. An appropriate cellulasefree, commercially available, yeast-lysing enzyme preparation fromCytophaga was identified. A freeze-drying step was identified as necessary to render the SSF yeast cells susceptible to enzymatic lysis. The method was applied to the analysis of cellulose and yeast-associated glucans in SSF residues from three pretreated feedstocks; hybrid poplar, switchgrass, and cornstover. Cellulose assays employing the lysing-enzyme preparation demonstrated relative errors up to 7.2% when yeast-associated glucans were not removed prior to analysis of SSF residues. Enzymatic lysis of SSF yeast cells may be viewed as a general preparatory procedure to be used prior to subsequent chemical and physical analysis of SSF residues. Oregon State University Agricultural Experiment Station Technical Publication Number 10977.     

Noncompetitive enzyme immunoassay for α-fetroprotein using flow injection chemiluminescence

A novel, direct noncompetitive flow injection enzyme immunoassay for α-fetoprotein (AFP) was developed by enhanced chemiluminescence detection. The method was based on off-line incubation of AFP and horseradish peroxidase (HRP)-labeled anti-AFP, and then trapping of the unbound enzyme conjugate by an immunoaffinity column filled with AFP-modified Sepharose. The immunocomplex formed in incubation passed through the column and then was directly detected by a postcolumn chemiluminescence technique. The optimal conditions for the immunoassay procedure and chemiluminescence detection were established. At a 1:10 dilution of enzyme conjugate solution, the linear range for chemiluminescence detection of AFP was from 2.0 to 75 ng/mL with a correlation coefficient of 0.993 and a coefficient of variation of 2.67% at 30 ng/mL. The detection limit was 0.5 ng/mL. This method was flexible, sensitive, and rapid. The immunoaffinity column of 200 μL could be repeatedly used 100 times without a single decrease. The whole assay time including the preincubation step was only 30 min for one sample.     

Purification of glutathione reductase from chicken liver and investigation of kinetic properties

Glutathione reductase was purified from chicken liver and some characteristics of the enzyme were investigated. The purification procedure was composed of four steps: preparation of homogenate, ammonium sulfate precipitation, 2′,5′-ADP Sepharose 4B affinity chromatography, and Sephadex G-200 gel filtration chromatography. Owing to the four consecutive procedures, the enzyme was purified 1714-fold, with a yield of 38%. Specific activity at the final step was 120 enzyme unit (EU)/mg of protein. The purified enzyme showed a single band on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weight of the enzyme was found to be 100 kDa by Sephadex G-200 gel filtration chromatography, and the subunit molecular weight was found to be 43 kDa by SDS-PAGE. Optimum pH, stable pH, optimum ionic strength, and optimum temperature were 7.0, 7.4, 0.75 M Tris-HCl buffer including 1 mM EDTA, and 50°C, respectively. K _M and V _max values for NADPH and glutathione disulfide (GSSG) substrates were also determined for the enzyme.     

Comparative Study of Polymeric Supports as the Base of Immobilisation of Chemically Modified Enzymes

One of the difficulties in using optical biosensors based on enzymatic reactions is the immobilisation of the enzyme involved in the determination. A detailed study has been carried out of the various polymeric supports which could provide a potential alternative for the immobilisation of chemically modified enzymes. Before immobilisation, the enzyme is attached by means of a covalent bond to a fluorescent probe, which is optically active in the visible region. The main advantage of this covalent bond is that it is possible to follow the enzymatic reaction by fluorescence without the need for immobilising any further reagent (reagentless biosensors). The results indicate that the most stable and reproducible polymeric supports are derived from polyacrylamide obtained by UV photopolymerisation. The experiments have been carried out using chemically modified glucose oxidase as the model enzyme. The films thus obtained have a lifetime of at least two months, an RSD of 9.2%, and a linear range of 300 to 2000 mg L⁻¹ of glucose. They are completely reversible by regeneration in an appropriate buffer solution.     

Biotinyl-Glucose-6-Phosphate Dehydrogenase Preparation,Kinetics, and Modulation by Avidin

The kinetics of free glucose-6-phosphate dehydrogenase (G-6-PDH), biotinylated G-6-PDH, and biotinylated G-6-PDH complexed with avidin were investigated. The kinetics of the free enzyme were consistent with a sequential rather than a ping-pong mechanism. The kinetics of the biotinylated enzyme were similar to that of the free enzyme, but the kinetic constants were different; theK _m value for NADP was halved, whereas theK _m for G-6-P decreased only slightly. In the presence of avidin, theK _m of biotinylated G-6-PDH for G-6-P nearly doubled whereas theK _m for NADP did not change significantly. Avidin complexed with biotinylated G-6-PDH inhibited the enzyme from acting. Based upon these reactions, it was possible to devise assays for either free biotin or free avidin using biotinylated G-6-PDH as the indicator enzyme. Concentrations of biotin between 40 and 60 mg/mL, or of 25–95 Μg/mL of avidin could be measured within 2 min through the use of biotinylated G-6-PDH.     

氯化血红素作为模拟酶荧光免疫分析乙肝表面抗原

乙肝表面抗原的测定在临床诊断上是一项很重要的指标．现在一般采用酶联吸附免疫分析技术测定，但是酶本身性质不稳定且价格昂贵、操作繁琐；更重要的是大分子的酶作为标记物，由于空间位阻效应而阻碍抗原-抗体的免疫反应．所以，用小分子催化剂代替大分子酶的研究显得日益重要[1，2]．近年来有关模拟酶在免疫分析中的应用已有报道[3，4]．本文提出了以氯化血红素作为辣根过氧化物酶（HRP）的模拟酶来标记抗体，以盐酸硫胺素（维生素B1）作为供氢体，成功地实现了乙肝表面抗原（HBsAg）的夹心法荧光免疫分析．测定范围是2．5～500ng／wel…     

Attachment of horseradish peroxidase (HRP) onto the poly(styrene/acrolein) latexes and onto their derivatives with amino groups on the surface; activity of immobilized enzyme

The polystyrene (P(S)), poly(styrene/acrolein) (P(SA)), and polyacrolein (P(A)) latexes, with varied fraction of polyacrolein in the surface layer (f _A=0, 0.50, 0.63, 0.84, 1.00), were used for the attachment of horseradish peroxidase. Surfaces of latexes were modified by reaction with ethylenediamine. In this step the aldehyde groups from polyacrolein were blocked and the primary amino groups were introduced. The carbohydrate portion of HRP was oxidized in the reaction leading to formation of aldehyde groups. The adsorption and covalent immobilization of HRP onto the P(S), P(SA), and P(A) latexes and of the oxidized HRP (HRP-OX) onto the modified latex particles, with amino groups on the surface (P(SA)-M and P(A)-M), were investigated. The activities of parent and oxidized HRP were compared with activities of the corresponding enzymes in solution. It has been found that whereas HRP is not suitable for the covalent immobilization on P(SA) latex and loses its activity after adsorption onto P(S) latex, HRP-OX can be adsorbed onto P(S) latex and is readily immobilized covalently onto the ethylenediamine modified P(SA) and P(A) latexes, retaining much of its former enzymatic reactivity.This work was supported by the KBN Grant 2 0624 91 01     

木霉No.183菌株木聚糖酶的研究

 筛选到一株木聚糖酶高产木霉菌株(No.183),研究了该菌株产木聚糖酶的液态发酵和粗酶液的酶学性质.结果表明,以麸皮和木聚糖为主要碳源,28℃,190r/min摇瓶培养时,木霉No.183菌株在接种后84h酶活最高,达到298.47U/mL.该木聚糖酶的最适反应温度为50℃,最适pH为该木聚糖酶在pH5～7和40℃以下时相对稳定.Ca²⁺,Zn²⁺和Cu²⁺对该木聚糖酶有较强的促进作用,Fe³⁺和Hg²⁺对该酶有较强的抑制作用.     

合系35号水稻Q酶基因的克隆

 支链淀粉含量是稻米品质的评价标准之一,控制支链淀粉合成的酶是淀粉分支酶.依据GenBank公布的日本水稻淀粉分支酶(Q酶)基因的cDNA序列合成相应引物,应用RT-PCR技术,SOE法成功地克隆到云南合系35号水稻Q酶基因的cDNA全长编码框2464bp.与日本水稻比较,合系35的Q酶基因有16个位点存在差异,并导致10个位点的氨基酸变化.