Abstract：
Enzymes are important drug targets and inhibition of enzymatic activity is an important therapeutic strategy. Enzyme assays measuring catalytic activity are utilized in both the discovery and development of new drugs. Colorimetric assays based on the release of 4-nitrophenol from substrates are commonly used. 4-Nitrophenol is only partly ionized to 4-nitrophenolate under typical assay conditions (pH 7-9) leading to under-estimation of product formation rates due to the much lower extinction coefficient of 4-nitrophenol compared to 4-nitrophenolate. Determination of 4-nitrophenol pKa values based on absorbance at 405 nm as a function of experimental pH values is reported, allowing for calculation of a corrected extinction coefficient at the assay pH. Characterization of inhibitor properties using steady-state enzyme kinetics is demonstrated using calf intestine alkaline phosphatase and 4-nitrophenyl phosphate as substrate at pH ∼8.2. The following kinetic parameters were determined: Km= 40±3 µM; Vmax= 72.8±1.2 µmolmin-1mg protein-1; kcat= 9.70±0.16 s-1; kcat/Km= 2.44±0.16 × 105 M-1s-1 (mean± SEM, N = 4). Sodium orthovanadate and EDTA were used as model inhibitors and the following pIC50 values were measured using dose-response curves: 6.61±0.08 and 3.07±0.03 (mean±SEM, N = 4). Rapid dilution experiments determined that inhibition was reversible for sodium orthovanadate and irreversible for EDTA. A Ki value for orthovanadate of 51±8 nM (mean±SEM, N = 3) was determined. Finally, data analysis and statistical design of experiments are discussed.
摘要：
酶是重要的药物靶标,抑制酶活性是重要的治疗策略。测量催化活性的酶测定法用于发现和开发新药物。通常使用基于从底物释放4-硝基苯酚的比色测定。在典型的测定条件(pH7-9)下,4-硝基苯酚仅仅部分离子化为4-硝基苯酚盐,由于4-硝基苯酚的消光系数与4-硝基苯酚盐相比低得多,导致产物形成速率的估计不足。报告了基于405nm处的吸光度作为实验pH值的函数的4-硝基苯酚pKa值的测定。允许在测定pH下计算校正的消光系数。使用小牛肠碱性磷酸酶和4-硝基苯基磷酸酯作为底物，在pH〜8.2时，证明了使用稳态酶动力学表征抑制剂特性。确定了以下动力学参数：Km=40±3µM；Vmax=72.8±1.2µmolmin-1mg蛋白-1；kcat=9.70±0.16s-1；kcat/Km=2.44±0.16×105M-1s-1（平均值±SEM，N=4)。将原钒酸钠和EDTA用作模型抑制剂，并使用剂量反应曲线测量以下pIC50值：6.61±0.08和3.07±0.03（平均值±SEM，N=4)。快速稀释实验确定，抑制对原钒酸钠是可逆的，对EDTA是不可逆的。原钒酸盐的Ki值为51±8nM(平均值±SEM，确定N=3)。最后,对实验的数据分析和统计设计进行了讨论。
