Simultaneous determination of mono- and poly(ADP-ri-bose) in vivo by tritium labelling and direct high-performance liquid chromatographic separation.

A microanalytical method for the determination of cellular mono-, oligo-and poly(ADP-ribose) has been developed that does not involve enzymatic degradation of oligomers to ribosyladenosine. The method consists of separation of protein-bound mono-, oligo- and poly(ADP-ribose) adducts from soluble nucleotides, followed by hydrolysis and quantitative isolation of AMP [derived from mono-(ADP-ribose)proteins], oligo- and poly(ADP-ribose) by boronate affinity chromatography and subsequent isolation of these nucleotides by HPLC. cis-Diols in AMP, oligo- and poly(ADP-ribose) are selectively oxidized by periodate, then reduced by [3H]borohydride. Conditions for the oxidation-reduction steps were optimized, and tritiated AMP, oligo- and poly(ADP-ribose) were quantitatively determined by radiochemical analysis of these components that were isolated by reversed-phase high-performance liquid chromatography. A 1-pmol ADP-ribose unit under standard conditions yields 2 X 10(3)-2.2 X 10(3) cpm 3H and this sensitivity can be amplified by increasing the specific radioactivity of [3H]borohydride.