Microheterogeneity of the mammalian high-mobility group proteins 14 and 17 investigated by reverse-phase...

Microheterogeneity within the HMG-14 and HMG-17 group 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 as a weak ion-pairing agent) which separate proteins primarily on the basis of differences in their overall hydrophobicities. Ion-pair RP-HPLC proves to be a fast and efficient means for separating multiple subspecies of both the HMG-14 and the -17 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 two different HMG-14 protein species (one major and one minor) and three different HMG-17 species (two major and one minor) can be resolved by ion-pair RP-HPLC. The identity and purity of these HMG-14 and -17 protein species were assayed by polyacrylamide gel electrophoresis and amino acid analysis. The amount of HMG protein microheterogeneity observed by RP-HPLC equals or exceeds that found for these proteins by other analytical techniques and the results suggest that this heterogeneity may be due to factors other than protein size or overall net charge variability.