Ning Da-liang, Wang Hui, Li Dong
State Key Joint Laboratory of Environmental Simulation and Pollution Control, Department of Environmental Science and Engineering, Tsinghua University, Beijing 100084, China.
Huan Jing Ke Xue. 2009 Aug 15;30(8):2485-90.
The induction and measurement of cytochrome P450 in white rot fungus Phanerochaete chrysosporium were studied in this work. The spectrophotometric results demonstrated that n-hexane was able to induce the fungal P450 to high level, which facilitated isolation and measurement of microsomal P450. The highest concentration of microsomal P450 could reach 140-160 pmol/mg after 6-h-induction by addition of 2 microL/mL hexane each hour, and the concentration of hexane and incubation time had significant effect on the induction of P450s. After effective induction, the method for isolation and measurement of microsomal P450 with CO difference spectrum was studied and the optimized method was obtained as followed. High-speed disperser and glass homogenizer were used to disrupt cells, which obtained higher amount of microsomal P450 than those from cells disrupted by glass homogenizer, ultrasonicator and bead-beater respectively. To record CO difference spectrum,the sample was bubbled with CO for 40 s at a rate of 3 mL/min (300 microL sample), and the reference cuvette was bubbled with N2 to the same extent. Then, the reducer sodium dithionite was added to a concentration 0.4 mol/L.
本研究对白腐真菌黄孢原毛平革菌中细胞色素P450的诱导及测定进行了研究。分光光度法结果表明，正己烷能够将真菌P450诱导至高水平，这有利于微粒体P450的分离和测定。通过每小时添加2 μL/mL己烷进行6小时诱导后，微粒体P450的最高浓度可达140 - 160 pmol/mg，且己烷浓度和孵育时间对P450的诱导有显著影响。有效诱导后，研究了用CO差光谱法分离和测定微粒体P450的方法，并获得了如下优化方法。使用高速分散器和玻璃匀浆器破碎细胞，与分别用玻璃匀浆器、超声破碎仪和珠磨仪破碎细胞相比，前者获得的微粒体P450量更高。为记录CO差光谱，以3 mL/min的流速（300 μL样品）向样品中通入CO 40 s，向参比比色皿中以相同程度通入N2。然后，加入连二亚硫酸钠使其浓度达到0.4 mol/L。
