Recordkeeping, Writing,
& Data Analysis


Microscope studies

Flagella experiment
Laboratory math
Blood fractionation
Gel electrophoresis
Protein gel analysis
Concepts/ theory
Keeping a lab notebook
Writing research papers
Dimensions & units
Using figures (graphs)
Examples of graphs
Experimental error
Representing error
Applying statistics
Principles of microscopy

Solutions & dilutions
Protein assays
Fractionation & centrifugation
Radioisotopes and detection


Mitochondria theory

Mitochondria in vitro

Additional topics


Selected Metabolic Poisons

Here is a list of poisons that can be used in the study of oxygen consumption by mitochondria, including sources and considerations for their use.

  • Electron transport inhibitors
    • Rotenone
    • Antimycin
    • Cyanide
    • Malonate (succinate dehydrogenase inhibitor)
  • Uncoupling agents
    • 2,4-Dinitrophenol (DNP)
    • Carbonyl cyanide p-[trifluoromethoxy]-phenyl-hydrazone (FCCP)
  • Oligomycin (inhibitor of oxidative phosphorylation)
With the exception of malonate and cyanide, these poisons are much more soluble in ethanol than in water. Adding even a small quantity of ethanol to an aqueous medium increases its capacity for oxygen. A Clark electrode detects such an increase as a temporary rise in oxygen content, followed by the steady state that would be expected after the addition of the agent. We refer to this 'blip' on the record as an ethanol artifact.

Electron transport inhibitors

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.


Rotenone is still used as an insecticide, but is not available for general use. It is toxic to wildlife and to humans as well as to insects. The location of inhibition by this competitive inhibitor of electron transport can be worked out by testing its ability to block respiration via the NADH versus succinate pathway.


The antimycin that we use in research was formerly known as antimycin A. The latter term has been dropped since only one antimycin is used in the literature. The binding site for antimycin can be narrowed considerably using combinations of substrates inlcuding succinate, NADH or glutamate, and the dye TMPD (N,N,N',N'-tetramethyl-p-phenylenediamine) along with ascorbic acid.


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.


Malonate (malonic acid) has long been known to inhibit cellular respiration. Among the key observations made in the 1930s investigations into the nature of cellular respiration was that the addition of fumarate, malate, or oxaloacetate to cell preparations resulted in the accumulation of succinate in the presence of malonate. Malonate is in fact a competitive inhibitor, and although we treat it as an inhibitor of electron transport it really is an enzyme inhibitor.

Uncoupling agents

Uncoupling is defined as a condition in which the rate of electron transport can no longer be regulated by an intact chemiosmotic gradient. The condition is differentiated from electron transport inhibition by the fact that in the latter case, bypassing the block can restore the gradient. In uncoupling, the electron transport system is uninhibited due to complete and irreversible dissipation of the chemiosmotic gradient.


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.

Carbonyl cyanide p-[rifluoromethoxyl]-phenyl-hydrozone (FCCP)

This agent is, in fact, a pure uncoupler. It acts as an ionophore, completely dissipating the chemiosmotic gradient, leaving the electron transport system uninhibited. It is also expensive.


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.

Copyright and Intended Use
Visitors: to ensure that your message is not mistaken for SPAM, please include the acronym "Bios211" in the subject line of e-mail communications
Created by David R. Caprette (caprette@rice.edu), Rice University 12 Dec 96
Updated 31 May 05