RESUMO
Hydrophilic-interaction liquid chromatography (HILIC) has recently been introduced as a highly efficient chromatographic technique for the separation of a wide range of solutes. The present work was performed with the aim of evaluating the potential utility of HILIC for the separation of postranslationally acetylated histones. The protein fractionations were generally achieved by using a weak cation-exchange column and an increasing sodium perchlorate gradient system in the presence of acetonitrile (70%, v/v) at pH 3.0. In combination with reversed-phase high-performance liquid chromatography (RP-HPLC) we have successfully separated various H2A variants and posttranslationally acetylated forms 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 results demonstrate that for the analysis and isolation of modified core histone variants HILIC provides a new and important alternative to traditional separation techniques and will be useful in studying the biological function of histone acetylation.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Histonas/análise , Acetilação , Ácidos/química , Animais , Butiratos/farmacologia , Ácido Butírico , Cromatografia por Troca Iônica , Detergentes/química , Eletroforese Capilar , Eletroforese em Gel de Poliacrilamida , Vírus da Leucemia Murina de Friend , Histonas/química , Histonas/efeitos dos fármacos , Leucemia Eritroblástica Aguda , Octoxinol/química , Espectrofotometria Ultravioleta , Propriedades de Superfície , Células Tumorais Cultivadas , Ureia/químicaRESUMO
The effects of different buffer concentrations and compositions on the elution order and separation of H1 histone subtypes and their phosphorylated modifications isolated from several species was studied using high-performance capillary electrophoresis (CE). Various cations and anions were tested in an untreated silica capillary and low pH buffers, in the presence of the dynamic coating agent hydroxypropylmethyl cellulose. It was found that the cations and anions of buffers have a remarkable influence on both the efficiency and the selectivity of protein separations. A triethylammonium methanephosphonate system proved efficacious for the separation of rat histone subtype H1c from H1e and a perchlorate/triethylammonium phosphate system for the analysis of chicken and mouse linker histones. CE provides an attractive alternative to high-performance liquid chromatography and conventional gel electrophoresis.
