Lerho M, Favazza M, Houssier C
Laboratoire de Chimie Macromoléculaire et Chimie Physique, Université de Liège, Sart-Tilman, Belgium.
J Biomol Struct Dyn. 1990 Jun;7(6):1301-19. doi: 10.1080/07391102.1990.10508567.
The interaction of histone H5 labelled with fluorescein isothiocyanate (FITC) with DNA has been studied by fluorescence titration, and diffusion-enhanced fluorescence energy transfer (DEFET) measurements with Tb(III) lanthanide chelates as donors. Analysis of the binding data by the model of Schwarz and Watanabe (J.Mol.Biol. 163, 467-484 (1983)) yielded a mean stoichiometry of 60 nucleotides per H5 molecule, independently of ionic strength, in the range of 3 to 300 mM NaCl, at very low DNA concentration (6 microM in mononucleotide). It ensues an approximate electroneutrality of the saturated complexes. Histone H5 molecules appeared to be clustered along the DNA lattice in clusters containing on average 3 to 4 H5 molecules separated by about 79 base pairs, at mid-saturation of the binding sites. The interaction process was found highly cooperative but the cooperativity parameter was also insensitive to ionic strength in the above range. DEFET experiments indicated an important decrease of accessibility of the FITC label to the TbHED3A and TbEDTA- chelates with ionic strength in the 0 to 100 mM NaCl range. In the presence of DNA, H5 appears already folded at low ionic strength so that the FITC probe is also not accessible to the donor chelate. The present study constitutes an indispensable preliminary step to further studies on the localization of histone H5 in condensed chromatin structures.
通过荧光滴定以及以铽（III）镧系螯合物作为供体的扩散增强荧光能量转移（DEFET）测量，研究了异硫氰酸荧光素（FITC）标记的组蛋白H5与DNA的相互作用。采用施瓦茨和渡边（《分子生物学杂志》163, 467 - 484 (1983)）的模型对结合数据进行分析，结果表明，在3至300 mM NaCl范围内，于极低DNA浓度（单核苷酸中为6 μM）下，每个H5分子的平均化学计量比为60个核苷酸，且与离子强度无关。由此得出饱和复合物近似呈电中性。在结合位点半饱和时，组蛋白H5分子似乎沿DNA晶格聚集，平均每个簇含有3至4个H5分子，彼此间隔约79个碱基对。发现相互作用过程具有高度协同性，但协同参数在上述离子强度范围内也对离子强度不敏感。DEFET实验表明，在0至100 mM NaCl范围内，随着离子强度增加，FITC标记物与TbHED3A和TbEDTA螯合物的可及性显著降低。在有DNA存在的情况下，H5在低离子强度下似乎已折叠，因此FITC探针也无法被供体螯合物接近。本研究是进一步研究组蛋白H5在浓缩染色质结构中定位的不可或缺的初步步骤。
