Fluorescence study of the interaction of calf thymus histone H1 with DNA S. N. Khrapunov, A. V. Sivolob and N. E. Kucherenko Biology Department, Kiev State University, Vladimirskaya 60, Kiev, USSR (Received 19 October 1983) Complexes of histone HI from calf thymus with high molecular weight DNA have been studied. Structural changes within a molecule of histone HI and its binding with DN A were registered over the fluorescence of a single residue of tyrosine in H1 whereas the changes in compaction of DNA were registered turbidimetrically. Association constants of the histone H1 globular domain with DNA were found on the basis of fluorescence measurements at different concentrations of salt and urea. It is shown that, at physiological ionic strength, compaction of DNA, folding of the histone HI globular domain and sharp weakening of the latter's binding with DNA take place. The DNA compaction does not depend on the presence of urea in solution. It is concluded that the histone HI globular domain is not involved in DNA compaction in chromatin. The role of various structural regions of histone HI in chromatin structure stabilization is discussed. Keywords: DNA; histoneH1; calfthymus; fluorescence spectroscopy; chromatin Introduction Histone H1 is the most important factor in chromatin compaction t'2, interacting with both link regions3 and nucleosomal DNA 4'5 as well as with core histones6. The mechanism of high-order packing of chromatin is not properly understood at present and therefore the study of interaction of histone H1 with DNA is of great interest. At physiological ionic strength, histone HI induces a compact ~,-form of DNA 7, resembling toroidal 'doughnuts's. This process is accompanied by aggregation a.9. It is known that a molecule of histone H 1 has a three-domain structure including an unfolded N- terminal region (approximately 40 amino acid residues), a folded globular domain (approximately 40 to 120 residues) and unfolded C-terminal region occupying almost the half the length of the amino acid chain and containing the most lysine residues 1°. The structure formation within the globular domain takes place with the increase of the ionic strength of the solution. According to the n.m.r, spectroscopy data, there exist variations in the interaction of different parts of the histone H1 molecule with DNA la. However, the molecular mechanisms of the histone H 1 interaction with DNA have not been studied in detail. In particular, the role of the histone H1 globular domain in the course of interaction with DNA is not clear. Fluorescence of a single tyrosine residue of the histone H 1 molecule (Tyr 72), being a part of its globular domain, is sensitive both to the formation of the histone H1 structure ~ 1 and, as previously shown, to its binding with DNA ~4. This work presents the study of the histone H1 intera~ction with DNA and the structure of its molecule within the DNA-histone complex by the method of fluorescence spectroscopy. Experimental Histone H1 was isolated from calf thymus according to the method described in Ref. 15, with additional purification by Big-Gel Acrilex P-60 (Reanal, Hungary). Electrophoresis was conducted according to the method described in Ref. 16 in the presence of sodium dodecyl sulphate (SDS). High molecular weight DNA from calf thymus (Worthington, USA) was used. The concentration of stock solutions was determined spectrophotometricaUy using molar absorption coefficients e275= 13001 tool- tcm- t for protein and e260 = 65001 tool - ~cm- 1 for DNA ~ 7. The complexes ofhistone H1 with DNA were prepared by direct mixing, gradually adding the protein in a buffer containing 5 mM Tris-HCl, pH 7.4, into the DNA solution in the same buffer. Fluorescence measurements were performed in a 5 x 5 mm thermostatically controlled cell at 20°C on a spectrofluorimeter equipped with grating mono- chromators in the excitation and emission channels. A mercury vapour DRK-120 lamp served as excitation source. Registration of emission was conducted in a pulse counting mode. Excitation wavelengths, 2e~,were 280, 265 and 233 nm, fluorescence was registered at 2era = 309 nm. The screening effect of DNA was taken into account according to different fluorescence intensity of the histone H 1 in 0.8 M NaCI in the presence and absence of DNA, because under such conditions histone H1 entirely dissociates from DNA 9'14. The solution turbidity was measured according to apparent absorbance at 360 nm in a 0.5-cm thick cell using an SF-26 spectrophotometer (USSR). The effect of light scattering upon the intensity of fluorescence was taken into account as described previously14. 0141-8130/84/040199-04503.00 © 1984Butterworth& Co. (Publishers)Ltd Int. J. Biol. Macromol., 1984, Vol 6, August 199