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

• Published in: Journal of Chromatography A 1986, Vol.359, p.275-284
• Main Authors: Hakam, Alaeddin, McLick, Jerome, Kun, Ernest
• Format: Article
• Language: English
• ISSN: 0021-9673
• DOI: 10.1016/0021-9673(86)80081-4

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 (18). A 1-pmol ADP-ribose unit under standard conditions yields 2 · 10 3–2.2 · 10 3 cpm 3H and this sensitivity can be amplified by increasing the specific radioactivity of [ 3H]borohydride.