乳酸脱氢酶的流动注射分析—电化学检测体系的研究

本文研究了L－乳酸盐＋NADLDH／→/←丙酮酸＋NADH＋H^ 的酶反应动力曲线及NADH的电化学性质与流动伏安检测法，建立了乳酸脱氢酶（LDH）的单泵单液路流动注射分析－电化学检测体系，可测定LDH的活性和浓度。     

Fluorescence of horse liver alcohol dehydrogenase using one- and two-photon excitation

We examined the steady-state and time-resolved emission of liver alcohol dehydrogenase resulting from one-photon and two-photon excitation. Previous studies with one-photon excitation revealed that the two nonidentical tryptophan residues display different emission spectra and decay times. The use of two-photon excitation resulted in similar emission spectra, multiexponential intensity decays, time-resolved emission spectra, and anisotropy decays as was observed for one-photon excitation. These results suggest that both nonidentical tryptophan residues are excited to a similar extent for one- and two-photon excitation. However, the limiting anisotropy (r ₀) with two-photon excitation from 585 to 610 nm is below 0.1 and appears distinct from that observed previously forN-acetyl-l-tryptophanamide.Abbreviations LADH liver alcohol dehydrogenase - -NAD⁺ -nicotinamide adenine dinucleotide - OPE one-photon excitation - OPIF one-photon induced fluorescence - TPE two-photon excitation - TCSPC time-correlated single photon counting - TPIF two-photon induced fluorescence     

新疆五种蝗虫的生化遗传学研究

应用同工酶技术,对隶属于两科五属的五种蝗虫的乳酸脱氢酶(LDH)同工酶、酯酶(EST)同工酶和苹果酸脱氢酶(MDH)同工酶进行了生化遗传学研究,共分析了7个基因位点,采用Nei(1972)的公式,计算各位点上等位基因的频率,求出标准遗传距离(D)和物种分化时间(t),并进行了聚类分析,得出以三点结论:1.五种蝗虫种间LDH,EST和MDH在基因位点的表达,各位点上等位基因的分布以及同一位点上各等位基因的频率三方面均具有明显差异。2.这五种蝗虫及所分析的7个基因位点,都表现出较高程度的基因分化和遗传多态现象。3.初步确定了这五种蝗虫种间的亲疏关系及物种分化时间。     

Construction and Characterization of Direct Electron Transfer-Type Continuous Glucose Monitoring System Employing Thermostable Glucose Dehydrogenase Complex

Abstract

We demonstrated a direct electron transfer-type enzyme electrode using thermostable FAD-glucose dehydrogenase (FADGDH) consisting of three distinct subunits (an FAD-containing catalytic subunit, a cytochrome subunit, and a chaperone-like subunit) and its application in developing a continuous glucose monitoring (CGM) system without a synthetic electron mediator. An FADGDH-immobilized electrode showed current signals according to glucose concentration in the absence of a synthetic electron mediator. The sensor containing the FADGDH complex showed a stable response for 72 h at 37°C. Furthermore, the CGM response was well fitted to the gradient change in the glucose concentration obtained from system calibration.     

Rapid Method for Differentiation of Carbonic Anhydrase I from Carbonic Anhydrase II Activity

ABSTRACT

A simple method for differentiation of carbonic anhydrase (CA) I, from CA II, is described based on using the nicotinates as specific CA I inhibitors. Nicotinates inhibit the activity of CA I activity; at a concentration of 5x10^?1M 100% inhibition is complete. But no effect on CA II activity is observed. In vivo administration of xanthinol nicotinate in doses of 20 mg/kg b.w. completely inhibited erythrocyte CA I activity. Association in ex vivo of methyl-nicotinate at a concentration of 5x10^?1M did not further modify erythocyte CA activity that had already been reduced by i.v. administration of xanthinol nicotinate. The nicotinate class could be used as a test for an accurate differentiation of CA I from CA II activity in vitro, in vivo and in ex vivo; by subtraction of I from total CA activity, one could also find the erythrocyte CA II activity. The ex vivo assays, using the test with nicotinates we call Nicosilvanil, would easily allow monitoring of CA I and II activity changes under physiological, pathological and experimental conditions, in response to various endogenous or therapeutical stimuli with either activating or inhibitory effects. Considering convenience, simplicity, and cost effectiveness of the reagents for the Nicosilvanil Test, this assay has the potential of being useful in routine analysis and differentiation of CA I from CA II activity in erythrocyte samples, as well as in other tissues and organs.     

LC‐MS/MS method for simultaneous analysis of uracil, 5,6‐dihydrouracil, 5‐fluorouracil and 5‐fluoro‐5,6‐dihydrouracil in human plasma for therapeutic drug monitoring and toxicity prediction in cancer patients

The chemotherapeutic drug 5‐fluorouracil (5‐FU) is widely used for treating solid tumors. Response to 5‐FU treatment is variable with 10–30% of patients experiencing serious toxicity partly explained by reduced activity of dihydropyrimidine dehydrogenase (DPD). DPD converts endogenous uracil (U) into 5,6‐dihydrouracil (UH₂), and analogously, 5‐FU into 5‐fluoro‐5,6‐dihydrouracil (5‐FUH₂). Combined quantification of U and UH₂ with 5‐FU and 5‐FUH₂ may provide a pre‐therapeutic assessment of DPD activity and further guide drug dosing during therapy. Here, we report the development of a liquid chromatography–tandem mass spectrometry assay for simultaneous quantification of U, UH₂, 5‐FU and 5‐FUH₂ in human plasma. Samples were prepared by liquid–liquid extraction with 10:1 ethyl acetate‐2‐propanol (v/v). The evaporated samples were reconstituted in 0.1% formic acid and 10 μL aliquots were injected into the HPLC system. Analyte separation was achieved on an Atlantis dC₁₈ column with a mobile phase consisting of 1.0 mm ammonium acetate, 0.5 mm formic acid and 3.3% methanol. Positively ionized analytes were detected by multiple reaction monitoring. The analytical response was linear in the range 0.01–10 μm for U, 0.1–10 μm for UH₂, 0.1–75 μm for 5‐FU and 0.75–75 μm for 5‐FUH₂, covering the expected concentration ranges in plasma. The method was validated following the FDA guidelines and applied to clinical samples obtained from ten 5‐FU‐treated colorectal cancer patients. The present method merges the analysis of 5‐FU pharmacokinetics and DPD activity into a single assay representing a valuable tool to improve the efficacy and safety of 5‐FU‐based chemotherapy. Copyright © 2012 John Wiley & Sons, Ltd.     

Direct electron transfer of PQQ-glucose dehydrogenase at modified carbon nanotubes electrodes

In order to establish efficient enzyme-electrode-contacts for the pyrroloquinoline quinone dependent glucose dehydrogenase (PQQ-GDH) different immobilisation strategies are investigated. Multi-walled carbon nanotubes (MWCNT) on gold electrodes are modified by chemical treatment and by (poly)-aniline derivatives. The electropolymerisation of methoxy-m-anilinesulfonic acid and m-aminobenzoic acid on the MWCNTs allows the covalent coupling of the PQQ-GDH. Such a poly-[ASA-ABA]/MWCNT/Au electrode can achieve current densities of up to 500 μA/cm² at a potential of 100 mV vs. Ag/AgCl. Furthermore investigations with small amounts of free PQQ indicate direct electron transfer between enzyme and electrode.