Microheterogeneity of the mammalian high mobility group (HMG) proteins 1 and 2 investigated by reverse-phase high...

Microheterogeneity within the high mobility group (HMG)-1 and HMG-2 groups of nonhistone chromatin proteins has been investigated using reverse-phase high-performance liquid chromatography (RP-HPLC) under conditions (acetonitrile elution with 0.1% trifluoroacetic acid (TFA) as the counter ion) which separate proteins primarily on the basis of differences in their overall hydrophobicity. RP-HPLC proved to be a fast and efficient means for separating multiple subspecies of both the HMG-1 and HMG-2 proteins from both crude nuclear extracts and from ion-exchange column "purified" protein samples obtained from different types of mammalian cell nuclei. In crude nuclear extracts at least eight different HMG-2 protein species (two major and six minor), but only one major HMG-1 species, could be resolved by RP-HPLC. Three of the minor HMG-2 protein species could be isolated in "pure" form from crude extracts in one RP-HPLC step whereas under the same conditions the two major HMG-2 peaks (as well as the other minor species) were contaminated with either HMG-1 or HMG-3 (a degradation product of HMG-1). In crude extracts the major HMG-1 fraction always seems to be contaminated with one of the HMG-2 subfractions. RP-HPLC analysis of apparently "pure" protein preparations isolated by ion-exchange chromatography techniques revealed that "pure" HMG-1 can be resolved into at least three different protein species and "pure" HMG-2 into at least four different species. Amino acid analyses of different resolvable forms of the HMG proteins were not inconsistent with the suggestion that at least some of these may be primary sequence variants of the individual proteins, but other possibilities also exist.