Redox potentials of the flavoprotein lactate oxidase.
Redox potentials of the flavoprotein lactate oxidase.
复制标题
黄素蛋白乳酸氧化酶的氧化还原电位。
作者：
Stankovich,M;Fox,B
方法对乳酸氧化酶进行三种实验。在所有实验中，基本的光谱电化学电池、光谱和电化学设备（Stankovich, 1980）以及光化学实验的方法（Choong & Massey, 1980）与前面描述的相同。然而，电极结构在以下方面得到了改进：(a)将原始设计的参考电极和辅助电极中的玻璃屑和琼脂替换为多孔的Vycor或“渴玻璃”（Dow Corning, Toledo， OH）。口渴的杯子是dr。R. Ramette，卡尔顿学院，诺斯菲尔德，明尼苏达州(b)最初用于储存参比电极和辅助电极的Na2S204氧洗涤溶液被5-deazariboflavin介导的甘氨酸光反应以还原形式维持的甲基紫胶所取代（Choong & Massey, 1980）。电量滴定。库仑滴定按前面描述的方法进行（Stankovich, 1980）。在这个实验中，只有MV，酶和缓冲液存在。根据EF10X在450 nm处的初始吸光度和EF10X在450 nm处公布的摩尔吸光度值计算酶的总浓度（Lockridge et al., 1972）。MV2+在可见光区域不吸收。由于浓度和体积是已知的，传递1等量电荷所需的库仑数可以由法拉第数计算出来。传递的还原当量数由电流积分决定。添加还原等价物后记录酶谱。绘制了530、450和370 nm处的吸光度与加入的还原等效物的数量的关系。如果酶电子转移发生在连续的单电子步骤中，这种类型的图将是线性的，在n= 1和n= 2处出现明显的中断。如果发生这种情况，则可以计算出单电子和双电子还原物质在任何波长下的摩尔吸收率以及所观察到的单电子还原物质的百分比。电流效率也可以计算，是总电流的百分比转移到感兴趣的物种。
Methods Three types of experiments were performed on lactate ox-idase. In all experiments the basic spectroelectrochemical cell, the spectral and electrochemical equipment (Stankovich, 1980), and the methodology of the photochemical experimental (Choong & Massey, 1980) were the same as described pre-viously. However, the electrode construction was improved in the following ways:(a) The glass frits and agar in the reference and auxiliary electrodes of the original design were replaced by porous Vycor or “thirsty glass”(Dow Corning, Toledo, OH). Thirsty glass was the gift ofDr. R. Ramette, Carleton College, Northfield, MN.(b) The Na2S204 oxygen scrubbing solution originally used for storing thereference and auxiliary electrodes was replaced by methylviologen main-tained in the reduced form by the 5-deazariboflavin-mediated glycine light reaction (Choong & Massey, 1980). Coulometric Titration. The coulometric titration was done as described previously (Stankovich, 1980). In thisexperiment only MV, enzyme, and buffer were present. The total con-centration of enzyme was calculated from the initial absorbance of EF10X at 450 nm and the published molar absorptivity value of EF10X at 450 nm (Lockridge et al., 1972). MV2+ does not absorb in the visible region. Since the concentration and volume were known, the number of coulombs required to transfer 1 equiv of charge could be calculated from Faradays number. The number of reducing equivalents transferred was determined by integration of current. Enzyme spectra were recorded after incremental additions of reducing equivalents. The absorbances at 530, 450, and 370 nm were plotted vs. number of reducing equivalents added. This type of plot will be linear with distinct breaks occurring at n= 1 and n= 2 if the enzyme electron transfers occur in sequential single electron steps. If this occurs, the molar absorptivity at any wavelength of both one-electron-and two-electron-reduced species can be calculated as well as the percentage of one-electron-reduced species observed. Current efficiency can also be calculated, being the percentage of the total current transferred which is transferred to the species of interest.