PROPIDIUM IODIDE AND THE THIOL-SPECIFIC REAGENT DACM AS A DYE PAIR FOR FLUORESCENCE RESONANCE ENERGY-TRANSFER ANALYSIS - AN APPLICATION TO MOUSE SPERM CHROMATIN

The dyes N-(-7-dimethylamino-4-methyl-coumarinyl) maleimide and propid ium iodide, specific for the thiol group and DNA, respectively were co nsidered as a donor-acceptor couple suitable for investigating ''in si tu'' the relative spatial distribution of DNA and protamines in mouse spermatozoa chromatin. The two dyes are characterized by favourable sp ectral properties, so that a simplified analytical procedure, based on the measurement of both donor and acceptor emission in double-stained samples, can be applied to evaluate the relative efficiency of the en ergy transfer process and its topological distribution. The results ob tained indicate that during the maturation process: 1) the basic arran gement of protamine-DNA complex does not undergo structure changes, an d 2) the oxidation of sulfhydryl to disulfide groups, resulting in chr omatin stabilization, first involves the protamine thiols spatially cl oser to DNA. Fluorescence energy transfer imaging suggests that chroma tin stabilization starts in the midportion of the sperm head, then spr eads towards the periphery. (C) 1994 Wiley-Liss, Inc.