Histone fractionation by high-performance liquid chromatography on cyanoalkylsilane (CN) reverse-phase columns

Journal Article · Fri Dec 31 23:00:00 EST 1982 · Anal. Biochem.; (United States)

DOI:https://doi.org/10.1016/0003-2697(83)90200-2· OSTI ID:6983185

Gurley, L R; Prentice, D A; Valdez, J G; Spall, W D

Previous work described conditions for the rapid fractionation of histones by high-performance liquid chromatography (HPLC) using a reverse-phase ..mu..Bondapak C/sub 18/ column. That procedure resolved the major classes of histones with one exception: the more hydrophobic H2A variant, (MHP)H2A, was not resolved from the H4 histone class. This report extends that work describing experiments using a ..mu..Bondapak CN column which better resolves the classes of histones from each other including the resolution of (MHP)H2A from the H4. In addition, the less hydrophobic H2A variant, (LHP)H2A, is partially resolved from the (MHP)H2A, and the less hydrophobic H3 variant, (LHP)H3, is resolved from the more hydrophobic H3 variant, (MHP)H3. Lower trifluoroacetic acid (TFA) concentrations (0.1%) in the eluting water/acetonitrile solvent were used with the CN column than were used with the C/sub 18/ column which increased the sensitivity of histone detection by ultraviolet absorption at 206 nm. Greater than 95% of the total (/sup 3/H)lysine-labeled protein applied to the CN column was eluted from the column. Contaminating nonhistone proteins were found to chromatograph in the region of histone elution. These were greatly reduced by isolating nuclei prior to histone preparation. The fractionation of the histones appears to be based on the hydrophobic properties of the proteins. The histone fractions (identified by their electrophoretic mobilities) were eluted from the CN column in the following order: H1, H2B, (LHP)H2A, (MHP)H2A, H4, (LHP)H3, and (MHP)H3. Phosphorylated and acetylated histone species were not resolved from their unmodified parental species.

• Research Organization: Los Alamos National Lab., NM
• OSTI ID: 6983185
• Journal Information: Anal. Biochem.; (United States), Journal Name: Anal. Biochem.; (United States) Vol. 131; ISSN ANBCA
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
CHROMATIN
CHROMATOGRAPHY
CHROMOSOMES
FRACTIONATION
HISTONES
HYDRIDES
HYDROGEN COMPOUNDS
HYDROGEN ISOTOPES
ISOTOPE APPLICATIONS
ISOTOPES
LABELLING
LIGHT NUCLEI
LIQUID COLUMN CHROMATOGRAPHY
NUCLEI
NUCLEOPROTEINS
NUCLEOSOMES
ODD-EVEN NUCLEI
ORGANIC COMPOUNDS
ORGANIC SILICON COMPOUNDS
PROTEINS
RADIOISOTOPES
SEPARATION PROCESSES
SILANES
SILICON COMPOUNDS
TRACER TECHNIQUES
TRITIUM
USES
YEARS LIVING RADIOISOTOPES