Reaction of cyanide with cytochrome aa3 in isolated perfused rat head in situ.
The reaction of cyanide with cytochrome aa3 in intact mitochondria is known to differ significantly from the reaction with the isolated enzyme. To examine the cyanide reaction with cytochrome aa3 in situ, we studied the spectral characteristics and the reaction kinetics of cyanide with reduced brain cytochrome aa3 in an isolated perfused rat head preparation. Anaesthetized rats underwent bilateral carotid-arterial cannulation. The head (skull intact, muscle removed) was perfused with a crystalloid solution containing Na2S2O4, and the animal was then decapitated. By means of reflectance spectrophotometry the reaction of cyanide with cytochrome aa3 was continuously monitored with the use of the 590 nm-575 nm, 610 nm-575 nm and 590 nm-610 nm wavelength pairs. We found that: the kinetics of the absorbance change at 590 nm and 610 nm were similar, with almost identical apparent rate constants, suggesting that these spectral changes are the results of the formation of a single complex; the difference spectrum obtained on addition of cyanide to the fully reduced preparation showed a peak at 588 nm and a trough at 610 nm, consistent with spectral characteristics of the cyanide-ferrocytochrome aa3 complex in isolated enzyme and isolated mitochondria in vitro; this observation underscores the accuracy of monitoring the effects of inhibitors of mitochondrial function on cytochrome redox reactions in situ; the half-maximal (K0.5) effect was approx. 50 microM, significantly lower than that in vitro. The lower apparent K0.5 for cyanide in this preparation in situ may be due to a difference in the pH of the two systems. This approach provides the means to study the inhibitors of mitochondrial function in intact brain under a physiological environment.
D000818:Animals; D001921:Brain / Q000187:drug effects / Q000201:enzymology*; D003486:Cyanides / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D003576:Electron Transport Complex IV / Q000378:metabolism*; D007700:Kinetics; D008297:Male; D010084:Oxidation-Reduction; D010477:Perfusion; D011190:Potassium Cyanide / Q000494:pharmacology*; D051381:Rats; D011919:Rats, Inbred Strains; D013053:Spectrophotometry
D003486:Cyanides; D003576:Electron Transport Complex IV; D011190:Potassium Cyanide