Kiehlbauch C C, Aboul-Ela N, Jacobson E L, Ringer D P, Jacobson M K
Department of Biochemistry & Molecular Biology, Texas College of Osteopathic Medicine, University of North Texas, Fort Worth 76107.
Anal Biochem. 1993 Jan;208(1):26-34. doi: 10.1006/abio.1993.1004.
Methods are described for the high resolution fractionation and characterization of ADP-ribose polymers. Polymers prepared in vitro using purified poly(ADP-ribose) polymerase were isolated free from interfering nucleic acids and salts using dihydroxyboronyl-Bio-Rex 70 chromatography and fractionated using anion exchange high-pressure liquid chromatography. The homogeneity of isolated polymer fractions was characterized by gel electrophoresis and polymer size was determined by analysis following enzymatic digestion to nucleosides. The method allows isolation of oligomers up to 50 mer as single species and larger polymers can be isolated free from oligomers according to size and branching frequency. The ability to isolate individual species of ADP-ribose polymers should prove useful for the study of the polymers and their noncovalent interactions with other components of chromatin. Microheterogeneity of individual oligomers was studied and shown to be due to differences at the protein proximal ends resulting from the chemical method of release of polymers from protein. The method also was applied to fractionate polymers generated in intact cultured mouse cells in response to treatment with the carcinogen N-methyl-N'-nitro-N-nitrosoguanidine.
本文描述了用于ADP-核糖聚合物的高分辨率分级分离和表征的方法。使用纯化的聚（ADP-核糖）聚合酶在体外制备的聚合物，通过二羟基硼酰基-Bio-Rex 70色谱法从干扰性核酸和盐中分离出来，并使用阴离子交换高压液相色谱法进行分级分离。通过凝胶电泳表征分离的聚合物级分的均一性，并通过酶促消化为核苷后的分析确定聚合物大小。该方法允许分离出长达50聚体的寡聚物作为单一物种，并且可以根据大小和分支频率从寡聚物中分离出更大的聚合物。分离单个ADP-核糖聚合物物种的能力对于研究聚合物及其与染色质其他成分的非共价相互作用应该是有用的。研究了单个寡聚物的微观异质性，并表明这是由于从蛋白质中释放聚合物的化学方法导致蛋白质近端的差异所致。该方法还应用于对完整培养的小鼠细胞中因致癌物N-甲基-N'-硝基-N-亚硝基胍处理而产生的聚合物进行分级分离。
