Sorouraddin Mohammad-Hossein, Fooladi Ebrahim, Naseri Abdolhossein, Rashidi Mohammad-Reza
Department of Analytical Chemistry, Faculty of Chemistry, Tabriz University, Tabriz, Iran.
J Biochem Biophys Methods. 2008 Apr 24;70(6):999-1005. doi: 10.1016/j.jbbm.2007.09.001. Epub 2007 Sep 18.
Although phenanthridine has been frequently used as a specific substrate for the assessment of aldehyde oxidase activity, the use of this method is questionable due to a lower limit of detection and its validity for kinetic studies. In the present study, a novel sensitive multivariate calibration method based on partial least squares (PLS) has been developed for the measurement of aldehyde oxidase activity using phenanthridine as a substrate. Phenanthridine and phenanthridinone binary mixtures were prepared in a dynamic linear range of 0.1-30.0 microM and the absorption spectra of the solutions were recorded in the range of 210-280 nm in Sorenson's phosphate buffer (pH 7.0) containing EDTA (0.1 mM). The optimized PLS calibration model was used to calculate the concentration of each chemical in the prediction set. Hepatic rat aldehyde oxidase was partially purified and the initial oxidation rates of different concentrations of phenanthridine were calculated using the PLS method. The values were used for calculating Michaelis-Menten constants from a Lineweaver-Burk double reciprocal plot of initial velocity against the substrate concentration. The limits of detection for phenanthridine and phenanthridinone were found to be 0.04+/-0.01 and 0.03+/-0.01 microM (mean+/-SD, n=5), respectively. Using this method, the Km value for the oxidation of phenanthridine was calculated as 1.72+/-0.09 microM (mean+/-SD, n=3). Thus, this study describes a novel spectrophotometric method that provides a suitable, sensitive and easily applicable means of measuring the kinetics of phenanthridine oxidation by aldehyde oxidase without the need for expensive instrumentation.
尽管菲啶经常被用作评估醛氧化酶活性的特异性底物，但由于其检测下限较低以及对动力学研究的有效性存疑，该方法的使用存在问题。在本研究中，已开发出一种基于偏最小二乘法（PLS）的新型灵敏多元校准方法，用于以菲啶为底物测量醛氧化酶活性。在0.1 - 30.0微摩尔的动态线性范围内制备菲啶和菲啶酮二元混合物，并在含有乙二胺四乙酸（0.1毫摩尔）的索伦森磷酸盐缓冲液（pH 7.0）中，于210 - 280纳米范围内记录溶液的吸收光谱。使用优化的PLS校准模型计算预测集中每种化学物质的浓度。对大鼠肝脏醛氧化酶进行部分纯化，并使用PLS方法计算不同浓度菲啶的初始氧化速率。这些值用于根据初始速度对底物浓度的Lineweaver - Burk双倒数图计算米氏常数。发现菲啶和菲啶酮的检测限分别为0.04±0.01和0.03±0.01微摩尔（平均值±标准差，n = 5）。使用该方法，计算出菲啶氧化的Km值为1.72±0.09微摩尔（平均值±标准差，n = 3）。因此，本研究描述了一种新型分光光度法，该方法提供了一种合适、灵敏且易于应用的手段，用于测量醛氧化酶氧化菲啶的动力学，而无需昂贵的仪器设备。
