Simultaneous determination of linear and branched residues in poly(ADP-ribose)

Methodology for the routine and simultaneous determination of the linear and branched residues of poly(ADP-ribose) is described. The main features of the procedure consist of the isolation of poly(ADP-ribose) by affinity chromatography; enzymatic digestion of the polymer to the unique nucleosides ribosyladenosine and diribosyladenosine which are derived from linear and branched residues, respectively; formation of fluorescent derivatives of ribosyladenosine and diribosyladenosine; and identification and quantification of these compounds by high-pressure liquid chromatography coupled with fluorescence detection. A variation on the methodology which allows the detection and quantification of ribosyladenosine and diribosyladenosine without formation of their fluorescent derivatives is also presented. Analyses of several cell lines for their capacity to synthesize poly(ADP-ribose) with a branched structure showed that the proportion of branched sites was constant (0.7-0.8%) in each of the cell lines.