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The in situ labeling of histone H3 in chromatin by a fluorescent probe
Abstract:
A sulfhydryl-specific fluorescent probe, N-3-pyrene maleimide, has been shown to label with high efficiency the sulfhydryl groups of histone H3 in nonsheared chromatin. The probe labels chromatin preparations obtained by mild homogenization or nuclease treatment of rat liver and mouse thymocyte, but not chick erythrocyte nuclei. Mononucleosomes from all nuclear preparations are labeled by the probe. The reaction is inhibited by prior reaction of the chromatin with N-ethyl maleimide. The reaction kinetics show fast and slow components representing reactions with cysteinyl sulfhydryl groups and lysyl epsilon-amino groups, respectively. Dissociation of the chromatin by urea (6 M) or sodium dodecyl sulfate (SDS) increases the fluorescence intensity (2-3 fold) and is maximal at approx. 0.01-0.02% (w/v) SDS. Histones extracted from the labelled chromatin show that approx. 80-90% of the label is associated with the histone fraction and column chromatography of this fraction shows that the label is primarily associated with histone H3. Labelling of the isolated histone fractions shows significant labelling only of histone H3. The intrinsic fluorescence of tryptophan is quenched by the labelled histone H3, but not by iodide, suggesting that non-histone (tryptophan-containing) proteins lie in close proximity to the labelled histone H3 but are not immediately accessible to external solvent. The labelled chromatin exhibits fluorescence anistropy, the anistropy parameter being 0.19 +/- 0.003 for chromatin, 0.05 +/- 0.01 for mononucleosomes and 0.0 for isolated histone H3. This demonstrates the restriction placed on the label's mobility by the chromatin fiber. The formation of a superhelix at 60-100 mM NaCl has been monitored with the probe. An increase in fluorescence intensity at 80 mM NaCl is observed with intact chromatin (but not H-1 depleted chromatin) followed by dissociation of the octamer in 1.50-2.0 M salt accompanied by a large increase in labelling.