High mobility group proteins HMG1 and -2 and histone H1 are structural components of chromatin. Previously, we reported that HMG1 interacts with H1 histone in a way that modulates the ability of H1 to condense DNA in vitro, suggesting that these proteins may act together in vivo to regulate locally the condensation state of chromatin, possibly affecting replication and/or transcription. Here we show that reduced (native) HMG1 binds to H1 cooperatively at pH 6.0 as a tetramer with a dissociation constant of 3.4 x 10(-8) M, and at pH 7.5 as a monomer with a dissociation constant less than 10(-9) M. Denaturation through oxidation of sulfhydryl groups has a strong effect on the interaction of HMG1 with H1 histone, suggesting that the reduced state of HMG1 is critical to its function. Oxidized HMG1 failed to bind H1 at pH 7.5, and its binding at pH 6 was biphasic; the first three (or two) molecules of H1 were bound with a dissociation constant of 2 x 10(-8) M with negative cooperativity, and the last one (or two) H1's were bound cooperatively with KD = 1.8 x 10(-7) M. Regulation of the pH or the concentration of some other ion may be used in vivo to alter the interactions between HMG1 and -2, H1 histone, and DNA.

Oxidized forms of non-histone chromosomal proteins high mobility group 1 (HMG1) and HMG2 were detected by high-pressure liquid chromatography of preparations stored at 4 degrees C for 1 day. The oxidized form of each was found to have two free sulfhydryl groups, while the freshly prepared native form of each contained four. The native, reduced state could be maintained during storage by the addition of ethylenediaminetetraacetic acid or reducing agents.

Fluorescence quenching was used to test the effect of pH changes on the binding of high-mobility group protein 1 (HMG1) to double-stranded and single-stranded DNA. At pH 7.5, the binding constant K for double-stranded DNA was 3 x 10(6) M-1, the binding site size n was 13, and the cooperativity factor q was 78, while at pH 6 the corresponding values were K = 12 x 10(6) M-1, n = 54, and q = 770. For the binding of HMG1 to single-stranded DNA at pH 7.5, the values were K = 2 x 10(6) M-1, n = 7, and q = 60, whereas at pH 6 they were K = 3 x 10(6) M-1, n = 14, and q = 440. Denaturation of HMG1 by oxidation of its sulfhydryl groups substantially affected the binding parameters. At pH 6, double-stranded DNA bound oxidized HMG1 with K = 6 x 10(6) M-1, n = 16, and q = 200, and single-stranded DNA bound with K = 3 x 10(6) M-1, n = 7, and q = 180. The sensitivity of the double-stranded DNA-HMG1 interaction to pH, along with an earlier report of a sharp optimum of binding at 140 mM NaCl, reveals a potential for in vivo regulation of the strength and mode of HMG1 binding by DNA through the action of analogous factors in the cellular milieu.