Raha C R, Gallagher C H, Shubik P, Peratt S
J Natl Cancer Inst. 1976 Jul;57(1):33-8. doi: 10.1093/jnci/57.1.33.
Incubation of 20,000 X g supernatant proteins of rat liver with benzo]a]pyrene (BP) and appropriate cofactors produced metabolites of BP covalently bound at the 4,5- position to protein. After being incubated, protein samples were freed from noncovalently bound metabolites and hydrolyzed chemically or both enzymatically and chemically. All steps were performed under minimum exposure to light and air; benzene extracts were prepared during hydrolysis. Chemical hydrolysis released the K-region metabolite BP-4,5-dihydrodiol (identified by UV and mass spectra) and BP. Hydrolysis with trypsin and pronase gave products with the characteristic UV spectrum of substituted chrysene. Chemical hydrolysis of these products released BP and BP-4,5-dihydrodiol.
将大鼠肝脏20,000×g上清液蛋白与苯并[a]芘（BP）及合适的辅助因子一起孵育，可产生 BP 在4,5-位与蛋白共价结合的代谢产物。孵育后，蛋白样品中非共价结合的代谢产物被去除，并进行化学水解，或同时进行酶解和化学水解。所有步骤均在最少的光照和空气暴露下进行；水解过程中制备苯提取物。化学水解释放出K-区域代谢产物BP-4,5-二氢二醇（通过紫外光谱和质谱鉴定）和BP。用胰蛋白酶和链霉蛋白酶水解得到具有取代屈特征紫外光谱的产物。这些产物的化学水解释放出BP和BP-4,5-二氢二醇。
