SEPARATION OF ACETYLATED CORE HISTONES BY HYDROPHILIC-INTERACTION LIQUID-CHROMATOGRAPHY

Hydrophilic-interaction liquid chromatography (HILIC) has recently bee n introduced as a highly efficient chromatographic technique for the s eparation of a wide range of solutes. The present work was performed w ith the aim of evaluating the potential utility of HILIC for the separ ation of posttranslationally acetylated histones. The protein fraction ations were generally achieved by using a weak cation-exchange column and an increasing sodium perchlorate gradient system in the presence o f acetonitrile (70%, v/v) at pH 3.0. In combination with reversed-phas e high-performance liquid chromatography (RP-HPLC) we have successfull y separated various H2A variants and posttranslationally acetylated fo rms of H2A variants and H4 proteins in very pure form. An unambiguous assignment of the histone fractions obtained was performed using high- performance capillary and acid-urea-Triton gel electrophoresis. Our re sults demonstrate that for the analysis and isolation of modified core histone variants HILIC provides a new and important alternative to tr aditional separation techniques and will be useful in studying the bio logical function of histone acetylation.