Separation of nonhistone high mobility group [HMG] from human lymphocytes by high performance liquid chromatography

ABSTRACT

The high mobility group [HMG] of nonhistone proteins have been investigated using two high performance liquid chromatographic techniques [HPLC]. Reversed-phase HPLC under conditions of 50 mM triethylamine adjusted to pH 2.2 with phosphoric acid [solvent A] and 95% acetonitrile in water [solvent B] was used to separate proteins primarily on the basis of differences in the overall hydrophobicity. Size exclusion HPLC under conditions of two different solvents [A, 0.1% trifluoroacetic acid TFA; B, 1.0% sodium dodecyl sulphate, SDS] was used to separate proteins. HMG proteins from human lymphocytes were separated into the HMG 1, HMG 2, HMG 14 and HMG 17 components. RP-HPLC is a proper method to resolve all the human lymphocyte HMG-proteins. Size exclusion HPLC was employed to resolve the HMG-protein subunits and determine their molecular weights. Ideal SE-HPLC is not capable of resolving HMG 1 from HMG 2 or HMG 14 from HMG 17 due to their molecular weight similarities. The purity of protein fractions were examined by acetic acid-urea-triton X-100 gel electrophoresis