Frasconi Marco, Rea Sara, Matricardi Pietro, Favero Gabriele, Mazzei Franco
Department of Chemistry and Drug Technologies, Sapienza University of Rome, P.le Aldo Moro, 5 00185 Rome, Italy.
Langmuir. 2009 Sep 15;25(18):11097-104. doi: 10.1021/la901245z.
A highly stable biological film was prepared by casting an aqueous dispersion of protein and composite hydrogel obtained from the polysaccharide Scleroglucan (Sclg) and borax as a cross-linking agent. Heme proteins, such as hemoglobin (Hb), myoglobin (Mb), and horseradish peroxidase (HRP), were chosen as model proteins to investigate the immobilized system. A pair of well-defined quasi-reversible redox peaks, characteristics of the protein heme FeII/FeIII redox couples, were obtained at the Sclg-borax/proteins films on pyrolytic graphite (PG) electrodes, as a consequence of the direct electron transfer between the protein and the PG electrode. A full characterization of the electron transfer kinetic was performed by opportunely modeling data obtained from cyclic voltammetry and square wave voltammetry experiments. The efficiency of our cross-linking approach was investigated by studying the influence of different borax groups percentage in the Sclg matrix, revealing the versatility of this hydrogel in the immobilization of redox proteins. The native conformation of the three heme proteins entrapped in the hydrogel films were proved to be unchanged, reflected by the unaltered Soret adsorption band and by the catalytic activity toward hydrogen peroxide (H2O2). The main kinetic parameters, such as the apparent Michaelis-Menten constant, for the electrocatalytic reaction were also evaluated. The peculiar characteristics of Sclg-borax matrix make it possible to find wide opportunities as proteins immobilizing agent for studies of direct electrochemistry and biosensors development.
通过浇铸由多糖硬葡聚糖（Sclg）和作为交联剂的硼砂获得的蛋白质和复合水凝胶的水分散体制备了一种高度稳定的生物膜。选择血红素蛋白，如血红蛋白（Hb）、肌红蛋白（Mb）和辣根过氧化物酶（HRP）作为模型蛋白来研究固定化系统。由于蛋白质与热解石墨（PG）电极之间的直接电子转移，在PG电极上的Sclg-硼砂/蛋白质膜上获得了一对明确的准可逆氧化还原峰，这是蛋白质血红素FeII/FeIII氧化还原对的特征。通过对循环伏安法和方波伏安法实验获得的数据进行适当建模，对电子转移动力学进行了全面表征。通过研究不同硼砂基团百分比在Sclg基质中的影响，研究了我们交联方法的效率，揭示了这种水凝胶在氧化还原蛋白固定化中的多功能性。水凝胶膜中包封的三种血红素蛋白的天然构象被证明没有改变，这通过未改变的Soret吸附带和对过氧化氢（H2O2）的催化活性得以体现。还评估了电催化反应的主要动力学参数，如表观米氏常数。Sclg-硼砂基质的独特特性使得其作为蛋白质固定剂在直接电化学研究和生物传感器开发中具有广阔的应用前景。
