& Data Analysis
Protein gel analysis
Keeping a lab notebook
Writing research papers
Dimensions & units
Using figures (graphs)
Examples of graphs
Principles of microscopy
Solutions & dilutions
Fractionation & centrifugation
Radioisotopes and detection
ETS inhibitors act by binding somewhere on the electron transport chain, literally preventing electrons from being passed from one carrier to the next. They all act specifically, that is, each inhibitor binds a particular carrier or complex in the ETS. Irreversible inhibition results in a complete stoppage of respiration via the blocked pathway. Competitive inhibition allows some oxygen consumption since a "trickle" of electrons can still pass through the blocked site. Although it allows some oxygen consumption, competitive inhibition prevents maintenance of a chemiosmotic gradient, thus the addition of ADP can have no effect on respiration.
Whatever the mechanism of inhibition, an electron transport inhibitor can block respiration specifically along the NADH pathway, along the succinate pathway, or along the pathway that is common to both routes of electron entry. Careful addition of inhibitors to mitochondria on specific substrates can reveal the sites of inhibition. Some combinations of inhibitors enable demonstration of alternative entry points to the electron transport system.
Cyanide is an extremely effective reversible inhibitor of cytochrome oxidase. A concentration of 1 mM KCN is sufficient to inhibit oxygen consumption by mitochondria from a vertebrate source by >98%. For a nominally 2 ml chamber, a convenient concentration for the stock solution would be 0.5M (20 µl produces a 2.5 mM final concentration).
Mitochondria from some sources have cyanide resistant pathways. KCN solutions are volatile, so that a dilute solution left open to the atmosphere will quickly lose its potency. Concentrations greater than 1 mM have been known to cause uncoupling. In the presence of TMPD we have seen a dramatic increase in oxygen consumption upon the addition of excess cyanide, using a Clark electrode. Indications were that a non-biological mechanism was responsible.
Cyanide is one of the most deadly compounds in a laboratory. Stocks of the dry chemical should be stored under lock and key. As we know from the Tylenol incidents of a number of years ago, a 500 mg capsule can hold enough cyanide to kill a person. Because of its volatility, exposure to fumes from large quantities is hazardous.
The compound 2,4-dinitrophenol (DNP) acts as a proton ionophore, that is, it binds protons on one side of a membrane, and being fat-soluble it drifts to the opposite side where it loses the protons. Actually, the associations/dissociations are random, but the probability of binding is greatest on the side of the membrane with greatest proton concentration, and least on the side with the lesser concentration. Thus, it is impossible to maintain a proton gradient with sufficient DNP in the system.
DNP is known to have mixed actions, that is, it produces other effects in addition to uncoupling. DNP gradually inhibits electron transport itself as it is incorporated into mitochondrial membranes. The effects appear to depend on concentration of DNP and of mitochondria, and vary from one preparation to the next.
Back in the 1930s DNP was touted as an effective diet pill. Indeed, the uncoupling of electron transport from ATP synthesis allows rapid oxidation of Krebs substrates, promoting the mobilization of carbohydrates and fats, since regulatory pathways are programmed to maintain concentrations of those substrates at set levels. Since the energy is lost as heat, biosynthesis is not promoted, and weight loss is dramatic. However, to quote Efraim Racker (A New Look at Mechanisms in Bioenergetics, Academic Press, 1976, p. 155), ..."the treatment eliminated not only the fat but also the patients,...This discouraged physicians for awhile..."
It is not a good idea to mess with cellular metabolism.
Oligomycin, an antibiotic, acts by binding ATP synthase in such a way as to block the proton channel. That is the mechanism by which oligomycin inhibits oxidative phosphorylation. Experimentally, oligomycin has no effect on state IV respiration, that is, it has no direct effect on electron transport or the chemiosmotic gradient. On the other hand oligomycin prevents state III respiration completely. To draw the conclusion that an agent is an inhibitor of ATP synthase (inhibitor of oxidative phosphorylation), the above conditions must be demonstrated experimentally and unequivocally.
It takes awhile for the effects of oligmycin to show up. Attempts to interrupt state III respiration by adding oligomycin may fail because of the delay.