12/2/2016

Metabolic poisons

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Selected Metabolic Poisons

organization of
the study
Here is a list of poisons that can be used in the study of oxygen consumption
polarography by mitochondria, including sources and considerations for their use.
calibrating
research paper
Electron transport inhibitors
Rotenone
Mitochondria theory
Antimycin
Cyanide
Malonate (succinate dehydrogenase inhibitor)
overview
Uncoupling agents
structure
2,4-Dinitrophenol (DNP)
Krebs reactions
Carbonyl cyanide p-[triuoromethoxy]-phenyl-hydrazone
electron
(FCCP)
transport
Oligomycin (inhibitor of oxidative phosphorylation)
the gradient
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
Mitochondria in
such an increase as a temporary rise in oxygen content, followed by the
vitro
steady state that would be expected after the addition of the agent. We refer to
this 'blip' on the record as an ethanol artifact.
preparation
fate of
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substrates
state IV
state III
metabolic
poisons
mitotraces
rationale
experiments
Additional topics
glossary of
terms
Hans Krebs
origin of
mitochondria
other functions

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 specically, 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 specically 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 specic substrates can
reveal the sites of inhibition. Some combinations of inhibitors enable
demonstration of alternative entry points to the electron transport system.

Rotenone
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.

Antimycin
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
Cyanide is an extremely effective reversible inhibitor of cytochrome oxidase.
A concentration of 1 mM KCN is sufcient 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
l produces a 2.5 mM nal 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
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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
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 dened 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.

2,4-Dinitrophenol
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 sufcient 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-[riuoromethoxyl]-phenyl-hydrozone

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(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
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

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Created by David R. Caprette (caprette@rice.edu), Rice University 12 Dec 96
Updated 31 May 05

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