Disaccharide modulation of the mitochondrial membrane fluidity changes induced by the membrane potential.

The influence of the medium composition on the dynamic properties of mitochondrial membranes on depolarization was studied by following the fluorescence anisotropy changes of mitochondria-bound 1,6-diphenyl-1,3,5-hexatriene (DPH) and hematoporphyrin (HP) as reporters, respectively, of lipid and protein regions. On collapse of the potential, the membrane fluidity increased in NaCl-, KCl-, and monosaccharide-based media and decreased in disaccharides. Infrared spectroscopy experiments suggested that disaccharides likely change water's structure and association on the membrane surface. These results indicate that disaccharides induce membrane perturbation, which may interfere in the study of structure-function correlation in biological membranes.

Implications of the generation of reactive oxygen species by photoactivated calcein for mitochondrial studies.

Calcein is a fluorescent probe that is widely used in studies of cell viability and mitochondrial function by microscopy fluorescence imaging. It was found to have a strong photosensitizing action that prevalently involves the generation of reactive oxygen species (ROS). The photooxidation properties of calcein in solution were studied in the presence of histidine and tryptophan as oxidizable substrates. The photodegradation of histidine was mainly mediated by singlet oxygen (1O2), as shown by the inhibitory effect of sodium azide, a specific 1O2 scavenger. On the other hand, mixed photosensitization mechanisms were present when tryptophan was used as the target of the calcein-stimulated photoprocess. In addition to 1O2, hydroxyl radicals and hydrogen peroxide were involved as reactive species, as shown by using mannitol and catalase as scavengers. The calcein-photosensitized alterations of mitochondria as a potential source of artifacts in confocal microscopy studies of cells were considered. Irradiation of isolated mitochondria with visible light (500-600 nm) in the presence of calcein induced opening of the permeability transition (PT) pore. The extent of the mitochondrial membrane photodamage, however, was modulated by the nature of the calcein environment. Thus, pore opening was triggered at short irradiation times and low dye concentrations when calcein was dissolved in the bulk medium. On the contrary, calcein concentrated in the matrix space was rather inefficient as photosensitizer even at concentrations 10 times higher than those present in the external medium.