High-performance liquid chromatography of in vitro synthesized poly(ADP-ribose) on ion-exchange columns, separation of oligomers of varying chain length and estimation of apparent branching.
Separated macromolecular fractions of in vitro synthesized poly(ADP-ribose) by liver nuclei were subjected to ion-exchange chromatography in a programmed high-performance liquid chromatographic elution system. The effects of ionic strength, pH and temperature on the separation of poly(ADP-ribose) chains were determined. Short chain oligomers (up to n = 11) were fractionated into individual components by baseline separation. Each fraction was analyzed for chain length. Trace amounts of Ado(P)Rib(P)Rib(P) found in phosphodiesterase digests were taken as indication of apparent branching. In phosphodiesterase digests of the shorter oligomers, besides traces of the above component, two other digestion products were also observed, presumably representing oligomer termini, one terminal fragment being dominant in short oligomers. Medium and long chain oligomers were partly resolved to individual components, and especially the long oligomers exhibited marked temperature dependent elution patterns. Apparent branching increased with increasing chain length up to about 3% for n = 44 and components presumably indicating termini diminished to mere traces. The adenine spectra of all fractions identified individual components.
D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D006868:Hydrolysis; D009702:Nucleoside Diphosphate Sugars / Q000302:isolation & purification*; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism; D011064:Poly Adenosine Diphosphate Ribose / Q000302:isolation & purification*; D013696:Temperature
D009702:Nucleoside Diphosphate Sugars; D011064:Poly Adenosine Diphosphate Ribose; D010727:Phosphoric Diester Hydrolases