title: Labelling of histone H5 and its interaction with DNA. 1. Histone H5 labelling with fluorescein isothiocyanate.
abstract: Histone H5 has been labelled with fluorescein isothiocyanate (FITC) with particular attention to the reaction conditions (pH, reaction time and input FITC/H5 molar ratio) and to the complete elimination of non-covalently bound dye. We preferred to use reaction conditions which yielded non-specific uniform labelling rather than specific alpha-NH2 terminal labelling, in order to obtain higher sensitivity in further studies dealing with the detection of perturbation at the binding sites of H5 on DNA. FITC-labelled H5 was further characterized by absorption and circular dichroism spectroscopy, and the fluorescein probe titrated in the 4-8 pH range. The structural integrity of H5 was found to be preserved after labelling. The positive electrostatic potential of the environment in which the FITC probe is embedded in the arginine/lysine-rich tails of H5 is believed to be responsible for the drop of pK of 1 unit found for H5-FITC as compared to free FITC. For the globular part of H5, the pK of covalently-bound FITC was only slightly lowered; this is a consequence of the much lower content in positively-charged amino-acid side chains in this region.
authors.affiliation: Laboratoire de Chimie Macromoléculaire et Chimie Physique, Université de Liège, Sart-Tilman, Belgium.
authors.forename: M
authors.initials: M
authors.lastname: Favazza
authors.forename: M
authors.initials: M
authors.lastname: Lerho
authors.forename: C
authors.initials: C
authors.lastname: Houssier
mesh_terms: D000818:Animals; D002645:Chickens; D002942:Circular Dichroism; D004247:DNA / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D016650:Fluorescein-5-isothiocyanate; D005452:Fluoresceins; D005456:Fluorescent Dyes; D006657:Histones / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D013861:Thiocyanates; D013997:Time Factors
chemical_list: D005452:Fluoresceins; D005456:Fluorescent Dyes; D006657:Histones; D013861:Thiocyanates; D004247:DNA; D016650:Fluorescein-5-isothiocyanate