Labelling of histone H5 and its interaction with DNA. 1. Histone H5 labelling with fluorescein isothiocyanate.
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.
Favazza
M
M
Laboratoire de Chimie Macromoléculaire et Chimie Physique, Université de Liège, Sart-Tilman, Belgium.
Lerho
M
M
Houssier
C
C
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
D005452:Fluoresceins; D005456:Fluorescent Dyes; D006657:Histones; D013861:Thiocyanates; D004247:DNA; D016650:Fluorescein-5-isothiocyanate