Effect of pH on the stability of hexokinase and glucose 6-phosphate dehydrogenase

Hexokinase (HK) and glucose 6-phosphate dehydrogenase (G6PDH) are important enzymes used in biochemical studies and in analytical methods. The stability of the enzymes can be affected by several variables, pH being one of them. The effect of pH on the stability of HK and G6PDH was evaluated in this work. Baker’s yeast cells were suspended in 50 mM Tris-HCl buffer (pH 7.5) containing 5.0 mM MgCl2, and submitted to disruption by agitation with glass beads and in the presence of protease inhibitors. The cell-free extract was obtained by centrifugation (2880g; 10 min), followed by dilution into the buffers: 0.1 M acetate-acetic acid (pH: 4.0, 4.5, 5.0, or 5.5), 0.1 M phosphate buffer (pH: 6.0, 6.5, or 7.0), and 0.1 M Tris-HCl buffer (pH: 7.5, 8.0, 8.5, 9.0 or 9.5). The residual activity of HK and G6PDH, expressed as μmol of NADPH formed per min, were measured through a period of buffer-enzyme contact from 0 to 51 h at 4°C. It was observed that up to 4 h both enzymes were stable in all buffers used. However, after 51 h HK was stable at pH 6.0 and 7.5, whereas G6PDH was stable at pH 7.0, 9.5, and between 4.5 and 5.5.     

Cyclometalated N,N-dimethylbenzylamine ruthenium(II) complexes [Ru(C6HRRR-o-CH2NMe2)(bpy)(RCN)2]PF6 for bioapplications: synthesis, characterization, crystal structures, redox properties, and reactivity toward PQQ-dependent glucose dehydrogenase

Cyclometalated derivatives of ring-substituted N,N-dimethylbenzylamines with controlled redox potentials as potent mediators of bioelectrochemical electron transport are reported. The cycloruthenation of R¹R²R³C₆H₂CH₂NMe₂ (R¹, R², R³ = H, Me, ^tBuO, MeO, NMe₂, F, CF₃, CN, NO₂) by [(η⁶-C₆H₆)RuCl(μ-Cl)]₂ in the presence of NaOH/KPF₆ in acetonitrile or pivalonitrile affords cyclometalated complexes [(η⁶-C₆H₆)Ru(C₆HR¹R²R³-o-CH₂NMe₂)(RCN)]PF₆ [R = Me (1) and R = CMe₃ (2)] in good yields. Reactions of complexes 1 and 2 with 2,2′-bipyridine (bpy) in acetonitrile or pivalonitrile result in dissociation of η⁶-bound benzene and the formation of [Ru(C₆HR¹R²R³-o-CH₂NMe₂)(bpy)(RCN)₂]PF₆ [R = Me (3) and R = CMe₃ (4)]. All new compounds have been fully characterized by mass spectrometry, ¹H/¹³C NMR, and IR spectroscopy. An X-ray crystal structural investigation of complex 1 (R¹/R²/R³ = H/H/H) and two complexes of type 3 (R¹/R²/R³ = MeO/H/H, MeO/MeO/H) has been performed. Acetonitrile ligands of 3 are mutually cis and the σ-bound carbon is trans to one of the bpy nitrogens. Measured by the cyclic voltammetry in MeOH as solvent, the redox potentials of complexes 3 for the Ru^II/III feature cover the range 320-720 mV (versus Ag/AgCl) and correlate linearly with the Hammett constants. Complexes 3 mediate efficiently the electron transport between the active site of PQQ-dependent glucose dehydrogenase (PQQ = pyrroloquinoline quinone) and a glassy carbon electrode. Determined by cyclic voltammetry the second order rate constant for the oxidation of the reduced (by d-glucose) enzyme active site by Ru^III derivative of 3 (R¹/R²/R³ = H) (generated electrochemically) is as high as 4.8 × 10⁷ M⁻¹ s⁻¹ at 25 °C and pH 7.     

Catalytic electrooxidation of NADH for dehydrogenase amperometric biosensors

The developments in the techniques of NADH catalytic oxidation relevant for incorporation in amperometric biosensors with dehydrogenase enzymes are reviewed with special emphasis in the years following 1990. The review stresses the direct electro-catalytic methods of NAD⁺ recycling as opposed to enzymatic regeneration of the coenzyme. These developments are viewed and evaluated from a mechanistic perspective of recycling of NADH to enzymatically active NAD⁺, and from the point of view of development of technologically useful reagentless dehydrogenase biosensors. An effort is made to propose a method for the standardization of evaluation of new mediating and direct coenzyme recycling schemes. A perspective is given for the requirements that have to be met for successful biosensor development incorporating dehydrogenase enzymes that open the analytical possibilities to a number of new analytes. The intrinsic limitations of the system are finally discussed and a view of the future of the field is presented.     

溶胶-凝胶固定化多酶催化二氧化碳转化为甲醇反应初探

 为了探索温室气体CO2的固定和利用的新途径，以正硅酸乙酯为\r\n前驱体，用改进的溶胶－凝胶法对甲酸脱氢酶、甲醛脱氢酶和乙醇脱氢\r\n酶进行了包埋共固定化，并以包埋的三种酶为催化剂，以还原型烟酰胺\r\n腺嘌呤二核苷酸（NADH）为电子供体，在低温低压下将CO2转化为甲醇\r\n．初步研究了反应温度、pH值、酶含量及NADH用量对甲醇收率的影响．\r\n实验结果表明，在37℃和pH7．0的条件下，甲醇的收率可达92．4％．\r\n由于酶空间构型的微小变化和空间位阻效应的存在，与液相酶反应结果\r\n相比，包埋后的酶活性略有降低．     

A new enzyme catalysis: 3,4-dioxidation of some aryl β-d-glycopyranosides by fungal pyranose dehydrogenase

Quinone-dependent pyranose dehydrogenase presents a new tool for versatile conversions of numerous carbohydrates to their di- and tricarbonyl derivatives. This enzyme purified from the basidiomycete Agaricus meleagris catalysed dioxidation of several aromatic β-d-glucopyranosides and a β-d-xylopyranoside into the corresponding 3,4-didehydro-β-d-aldopyranosides (β-d-aldopyranosid-3,4-diuloses) in high yields, typically >80% for 4-nitrophenyl glycosides. These new compounds were doubly hydrated in aqueous solution. According to in situ NMR investigations, the reaction intermediates were the corresponding 3- and 4-dehydro compounds. The analogous anomeric α-glycosides underwent one-step oxidation only at C-3 to 3-dehydro-α-d-aldopyranosides (α-d-pyranosid-3-uloses).     

Liquid—liquid extraction of lactate dehydrogenase from muscle using polymer-bound triazine dyes

An extract from porcine muscle containing soluble enzymes has been partitioned between the two liquid phases of an aqueous, biphasic system consisting of dextran, polyethylene glycol, and water. The influence of polymer-bound triazine dyes (Cibacron blue F3G-A and Procion yellow HE-3G) on the partition of lactate dehydrogenase and total protein was studied. The effects of pH and concentrations of polymers and buffer on this so-called affinity partitioning were also determined. The two-phase systems were used in extraction procedures for purification of lactate dehydrogenase to a specific activity of 456–494 U (7.6–8.4 μkat) per mg protein. The use of these systems for extraction of enzymes in technical scale is discussed.     

尼古丁对乳酸脱氢酶活性的影响

用计时电流法成功地研究了尼古丁对乳酸脱氢酶活性的影响,测定了尼古丁存在与否对乳酸脱氢酶酶促反应的初速度V0、酶促反应最大反应初速度Vm及米氏常数Km。实验结果表明尼古丁对乳酸脱氢酶的活性有很大影响,但在高浓度的NADH存在下,尼古丁对乳酸脱氢酶活性的影响将得到大大降低。     

电分析化学研究锌在乳酸脱氨酶和谷氨酸脱氢酶体系中的作用

酶是生物催化剂，在数以千计的酶中，第一大类以烟酰胺腺嘌呤核苷酸（ＮＡＤ／ＮＡＤＨ）为辅酶的多种氧化－还原酶需要金属离子作为辅助因子而发生独特的催化功能。其中含锌的醇脱氨酶（ＡＤＨ）是目前研究得最多的一种。本文用电化学分析法研究稀土离子对乳酸脱氢酶（ＬＤＨ）和谷氨酸脱氢酶（ＧＤＨ）两体系的作用时也探讨了Ｚｎ２＋在此两体系中的作用。结果指出在ＬＤＨ体系中不需要 Ｚｎ２＋；在 ＧＤＨ（牛肝中提取）体系中， Ｚｎ２＋是一种强抑制剂，Ｅｕ３＋可以缓解它的抑制作用，但是在黄瓜根系提取的粗ＧＤＨ体系中，Ｚｎ２＋必需存在于反介质中起激活酶的催化作用。     

Micro determination of cortisol and cortisone in umbilical cord blood by chemiluminescent high‐performance liquid chromatography

A simple, sensitive and specific chemiluminescent high‐performance liquid chromatography method, based on the luminol reaction, for determination of serum cortisol and cortisone, was established. In infants, placental 11β‐hydroxysteroid dehydrogenase type 2 enzyme (11β‐HSD2) activity may affect adrenal function early after birth. The cortisol–cortisone ratio of serum concentrations in umbilical cord blood is an indicator of placental 11β‐HSD2 activity. The optimum conditions for the luminol reaction were determined to be 1.5 mM luminol, 0.6 M sodium hydroxide, 0.15 mm potassium hexacyanoferrate(III) and 200 mM potassium hexacyanoferrate (II). The calibration curves for cortisol and cortisone exhibited good linearity. The correlation coefficients of the calibration curves were 0.996. The intra‐ and inter‐day precisions were in the ranges: cortisol 7.0–12.2 and 4.4–9.2%, cortisone 5.3–7.0 and 6.2–9.9%. The recoveries of these steroids were in the ranges: cortisol 97–105%, cortisone 94–102%. The limits of detection were as follows: cortisol, 0.17 μg/dl; cortisone 0.15 μg/dl. This assay could be successfully applied to determination of the cortisol–cortiosone ratio of serum concentrations in umbilical cord bloods. Copyright © 2009 John Wiley & Sons, Ltd.