Separation of poly(ADP-ribose) by high-performance liquid chromatography

Area of Science:

• Biochemistry
• Molecular Biology

Background:

• Poly(ADP-ribose) (PAR) is a crucial post-translational modification involved in DNA repair and gene regulation.
• Understanding PAR synthesis and detection is vital for studying cellular stress responses.

Purpose of the Study:

• To develop and validate a method for the in vitro synthesis of poly(ADP-ribose).
• To establish a reliable technique for the isolation and quantification of poly(ADP-ribose) from protein adducts.

Main Methods:

• In vitro synthesis of poly(ADP-ribose) using liver nuclei and NAD.
• Isolation of protein-poly(ADP-ribose) adducts.
• Hydrolysis (base or proteinase K) to release free polymers.
• Quantification using reversed-phase chromatography (Ultrasphere ODS 5-micron column).
• Detection of polymer modification via oxidation and borotritiation labeling.

Main Results:

• Successful in vitro synthesis of poly(ADP-ribose) homopolymers.
• Effective separation of free poly(ADP-ribose) from NAD, ADP-ribose, AMP, and adenosine.
• Demonstrated quantitative determination of poly(ADP-ribose) using chromatography.
• Confirmed that polymer modification (oxidation, labeling) is detectable.

Conclusions:

• The described method allows for the efficient synthesis and accurate quantification of poly(ADP-ribose).
• This technique provides a valuable tool for biochemical and molecular biology research involving poly(ADP-ribose).