[50] Determination of in Vivo levels of polymeric and monomeric ADP-ribose by fluorescence methods

This chapter describes the determination of in vivo levels of polymeric and monomeric ADP-ribose by fluorescence methods. Two key features that are common to both methods and are crucial in providing the necessary selectivity and sensitivity include the utilization of immobilized boronate resins to selectively and quantitatively adsorb polymeric or monomeric ADP-ribose from cell or tissue extracts and the conversion of adenine-containing compounds to highly fluorescent 1, N6-etheno derivatives which can be quantified at the picomole level. For polymeric ADP-ribose, the adenine-containing compounds are formed by the enzymatic hydrolysis of the polymer to generate unique adenosine derivatives from all internal residues. For monomeric ADP-ribose, the method involves chemical release of intact ADP-ribose residues from protein and quantification following conversion to the 1, N6-etheno(ADP-ribose). The assay for measurement of polymeric ADP-ribose is designed for up to 108 tissue culture cells or for up to 1.3 g (wet weight) of tissue. A real difficulty with regard to the quantification of monomeric ADP-ribose residues covalently bound to proteins is the limited knowledge of the chemical nature of the linkages that exists in vivo. Enzymes from eukaryotic sources have been purified that can catalyze the covalent attachment of single ADP-ribosyl residues to acceptor proteins via N-glycosylic linkages to the guanidino group of arginine residues.