Battaglini F, Koutroumanis M, English A M, Mikkelsen S R
Department of Chemistry and Biochemistry, Concordia University, Montreal, Quebec, Canada.
Bioconjug Chem. 1994 Sep-Oct;5(5):430-5. doi: 10.1021/bc00029a009.
The bimolecular rate constants for the reactions of five organic two-electron redox mediators with reduced glucose oxidase (GOx) were determined by measuring voltammetric electrocatalytic currents at glassy carbon electrodes in the presence of excess glucose under anaerobic conditions. The mediators studied were thionine, brilliant cresyl blue, azure A, daunomycin, and dopamine, and the bimolecular rate constants for electron transfer between GOx and the oxidized mediator (M-1 s-1) are 1.6 x 10(4), 4.0 x 10(2), 9.8 x 10(2), 9.0 x 10(3), and 1.2 x 10(6), respectively. GOx was covalently derivatized using 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide and N-hydroxysulfosuccinimide to form amide bonds between the aliphatic primary amine groups on daunomycin and dopamine and carboxylate side chains of aspartate and glutamate residues. Derivatives with 2.5 +/- 0.1 daunomycin groups and 4 +/- 1 dopamine groups were obtained, with activities of 50% and 75%, respectively, relative to native GOx in a dye-peroxidase assay. Although the daunomycin derivative did not show measurable intramolecular electron-transfer rates, the dopamine derivative rapidly transfers electrons from active-site FADH2 groups to the oxidized (quinone) form of dopamine. Because the heterogeneous oxidation of dopamine is relatively slow, the currents measured at +0.75 V vs Ag/AgCl were not at their limiting (plateau) values, and only a minimum value of the intramolecular rate constant (4.5 s-1) could be determined. This value is > 20 times larger than values obtained for GOx-ferrocene derivatives in which surface lysine residues were covalently modified using identical coupling reagents and similar reaction conditions. This work shows that targeting GOx carboxylate groups with electron-transfer mediators may represent a promising approach to the design of reagentless glucose biosensors.
在厌氧条件下，通过测量玻碳电极上过量葡萄糖存在时的伏安电催化电流，测定了五种有机双电子氧化还原介质与还原型葡萄糖氧化酶（GOx）反应的双分子速率常数。所研究的介质为亚甲蓝、灿烂甲酚蓝、天青A、柔红霉素和多巴胺，GOx与氧化态介质之间的电子转移双分子速率常数（M⁻¹ s⁻¹）分别为1.6×10⁴、4.0×10²、9.8×10²、9.0×10³和1.2×10⁶。使用1-乙基-3-[3-(二甲氨基)丙基]碳二亚胺和N-羟基磺基琥珀酰亚胺对GOx进行共价衍生化，以在柔红霉素和多巴胺上的脂肪族伯胺基团与天冬氨酸和谷氨酸残基的羧酸盐侧链之间形成酰胺键。得到了具有2.5±0.1个柔红霉素基团和4±1个多巴胺基团的衍生物，在染料过氧化物酶测定中，其活性分别相对于天然GOx为50%和75%。尽管柔红霉素衍生物未显示出可测量的分子内电子转移速率，但多巴胺衍生物能迅速将电子从活性位点FADH₂基团转移至氧化态（醌）形式的多巴胺。由于多巴胺的异相氧化相对较慢，相对于Ag/AgCl在+0.75 V时测量的电流未达到其极限（平台）值，仅能确定分子内速率常数的最小值（4.5 s⁻¹）。该值比使用相同偶联试剂和类似反应条件对表面赖氨酸残基进行共价修饰的GOx-二茂铁衍生物所获得的值大20倍以上。这项工作表明用电子转移介质靶向GOx羧酸盐基团可能是设计无试剂葡萄糖生物传感器的一种有前景的方法。
