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Lucigenin-dependent chemiluminescence together with 2-[4-iodophenyl]-3-[4-nitrophenyl]-5-[2,4-disulfophenyl]-2H tetrazolium monosodium salt (WST-1) reduction can be detected following addition of NADH to many cell types, including human sperm suspensions. Although many reports suggest that such a phenomenon is due to reactive oxygen species production, other oxygen detecting metabolite probes, such as MCLA and luminol, do not produce a chemiluminescent signal in this model system. The enzyme responsible for NADH-dependent lucigenin chemiluminescence was purified and identified as cytochrome-b5 reductase. In support of this concept, COS-7 cells overexpressing cytochrome-b5 reductase displayed at least a 3-fold increase in the previously mentioned activity compared with mock-transfected cells. Fractions containing cytochrome-b5 reductase were capable of inducing both lucigenin-dependent chemiluminescence and WST-1 reduction. Oxygen radicals clearly did not mediate the cytochrome b5-mediated activation of these probes in vitro since neither luminol nor MCLA gave a chemiluminescence response in the presence of the enzyme and the cofactor NADH. These results emphasize the importance of the direct NADH-dependent reduction of these putative superoxide-sensitive probes by cytochrome-b5 reductase even though this enzyme does not, on its own accord, produce reactive oxygen species.

The ability of 5 streptomycetous species synthesizing multicomponent antibiotic to produce the antibiotic and water-soluble pigment and to form the aerial mycelium in the presence of acridine dyes was studied. It was found that the character of the produced complex changed, when acridine dyes were added to the medium under conditions not affecting the culture growth and the temperature was elevated. Colonies deficient with respect to formation of the aerial mycelium and with changed pigment and antibiotic production were detected in the monospore cultures of the streptomycetes treated with acridine dyes, when the spore survival was equal to 100 percent, the frequency of the colonies being about 40 per cent.

The kinetics of the photodynamic desactivation of lysozyme in presence of acridine orange as the sensitizer have been investigated in detail varying oxygen, protein, dye concentration, ionic strength and pH value. The kinetics can be approximately described as an over all pseudo-first-order rate process. Changing the solvent from water to D2O or by quenching experiments in presence of azide ions it could be shown that the desactivation of lysozyme is caused exclusively by singlet oxygen. The excited oxygen occurs via the triplet state of the dye with a rate constant considerably lower than that to be expected for a diffusionally controlled reaction. Singlet oxygen react chemically (desactivation, k=2.9 X 10(7) m(-1) sec(-1)) and physically (quenching process, k=4.1 X 10(8) m(-1) sec(-1)) with the enzyme. The kinetical analysis shows that additional chemical reaction between singlet oxygen and lysozyme would have only little influence on the kinetics of the desactivation as long as their products would be enzymatically active and their kinetical constants would be less than about 1 X 10(8) m(-1) sec(-1)).

Single giant axons from the lobster circumesophageal connective were studied using the sucrose gap voltage-clamp technique. The axon area in the gap was bathed in acridine orange for several minutes and then rinsed for several minutes. Subsequent illumination resulted in progressive prolongation of electrically stimulated action potentials to durations of 150 msec. The prolongation was accompanied by an increase in threshold. Currents in voltage clamp were altered such that transient current inactivation was greatly slowed. The turn on of transient current was somewhat slowed, the voltage at which peak transient current could be obtained was shifted to more positive internal potentials, and transient current at all potentials was decreased. Steady-state current was similarly affected. Low calcium following illumination partially counteracted some of the changes, but not the slowing of inactivation. Low calcium increased the duration of prolonged action potentials. Selective alteration of parameters in the Hodgkin-Huxley equations brought about a qualitative match between computations and data.