Evaluación de Oferta y Demanda de Agua:

In the strictest sense a "free radical" is defined as "any chemical species capable of independent existence that contains one or more unpaired electrons" The unpaired electron may render the species highly reactive. This broad definition of a free radical includes the hydrogen atom, most transition metals and the oxygen molecule which has 2 unpaired electrons each located in a different n* antibonding orbitaP®. Oxygen does not behave as a highly reactive free radical because it's two unpaired electrons are located in differing 7i*2p orbitals and have parallel spins (ie they have the same spin quantum number).

Hence if oxygen attempts to oxidise another atom or molecule by accepting a pair of electrons from it, both of the new electrons must have parallel spins so as to fit into the vacant spaces in the n* orbitals. A pair of electrons in a molecular orbit will

normally have antiparallel spins. This imposes restrictions on the oxidations by molecular 0% and tends to make It accept its electrons one at a time (Figure 1,5), so slowing its reaction rate with non radical species.

o ‘2p

O

o

o

Tr'2p

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TT2p

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o 2p

©

©

©

0 ‘2s

©

©

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G 2s

©

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G*1s

©

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a 1s

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Ground state

Oa

Superoxide

Oa

Peroxide Ion

Oa®-

Figure 1,5 Electron bonding configurations in the diatomic oxygen molecule

A number of important molecular species may be generated during the reduction of molecular Og to water as illustrated in figure 1,6.

If a single electron is accepted by the ground state Og molecule, it must enter one of the 7Ü* antibonding orbitals, and the product formed is the superoxide radical.

Superoxide is formed in nearly all aerobic cells. One important source of superoxide is the respiratory burst of activated phagocytes (which include macrophages, neutrophils, eosinophils and monocytes). Addition of a second electron to the superoxide radical produces the peroxide ion which is not a free radical because it possesses no unpaired electrons. The pKa of the peroxide anion (Og^ ) favours its protonation at physiological pH to form hydrogen peroxide. The importance of HgOg lies in its water solubility and ability to diffuse comparatively long distances in biological systems before decomposing (especially in the

e

H

superoxide

O 2 * -

anlon

peroxide

anion

0 , 2

HO,*

H2O

2

perhydroxyl

radical

hydrogen

peroxide

hydroxyl

radical

hydroxyl

anion

H

OH* + OH

H

OH

HjO

Figure 1,6 Reduction of molecular oxygen to water by the sequential addition of 4 electrons presence of transition metal ions - vide infra) to form the extremely reactive hydroxyl radical (•OH). The •OH radical reacts with extremely high rate constants with almost every type of molecule found in living cells including sugars, amino acids, phospholipids, DMA bases and organic acids^®.

Mitochondrial cytochrome c oxidase, which accounts for most of the oxygen consumed by respiring cells, reduces oxygen to water with the release of only small amounts of superoxide (•Og ) or peroxide^^. This minimises the problem of •O2' toxicity by largely avoiding its production^®. There are several enzyme

reactions which produce larger amounts of •Oj'. For example xanthine oxidase and aldehyde dehydrogenase produce substantial amounts of Og' as do enzymes within phagocytes during the killing of engulfed microorganisms^® . Whilst there is evidence to suggest that superoxide itself is able to damage membranes®® and kill cells®\ there is little doubt that the production of •OH from the superoxide radical and the peroxide ion, usually in the presence of transition metal ions is a far more potent oxidant.

The hydroxyl radical Is amongst the most reactive species found in biological systems. Reactions of •OH may be divided into hydrogen abstraction, electron transfer (accepting or donating), and addition reactions^^. These reactions serve to illustrate an important point in radical chemistry - that reaction of a free radical with a non radical species often results in the generation of a different free radical which may be more or less reactive. Given that •OH is highly reactive, then the radical formed by its reaction with a molecular species is likely to be less reactive.

Transition metals contain an unpaired electron in the 3d orbital and exhibit a variable valency which allows them to undergo changes in oxidation state involving one electron transfer with comparative ease (eg. iron II, iron III, and iron IV). Hence these metals are often found at the centre of oxidases and oxygenases in biological systems because of their ability to accept and donate electrons in an energetically favourable manner. Copper also shares this ability, although it is not a transition metal. In its unionised form its 3d orbitals are full and there is a single electron in a 4s orbital. However unionised copper it readily forms Cu^" by the removal of two electrons (one from the 4s orbital and one from the 3d) and its valency is able to vary between Cu" and Cu^" by the filling and emptying of the remaining "space" in the 3d orbital (figure 1,7). This readiness to participate in redox reactions means that the transition metals may, under appropriate conditions, lead to the generation of highly reactive oxidising species following reactions with peroxides and superoxides^^.

The ability of a mixture of an iron (II) salt and hydrogen peroxide to form the •OH radical was noted as long ago as 1894 by Fenton^^ and this reaction now bears his name. The "Fenton reaction" is shown below;

Copper atom

Copper (I) Ion

Copper (II) Ion

3d

t t t t

tt n

ti Î

Figure 1,7 Electron outer orbitals of copper in atomic and ionic forms

4s

Î

Traces of can react further with HoO,:

Fe"" + H2O2 ^ Fe"" + *0,* + H+

Copper (I) salts can react with hydrogen peroxide in much the same way with a higher rate constant;

Cu^ + H2O2 -> Cu^" + •OH + OH'

In many systems which generate *0 2' , scavengers of the hydroxyl radical, *0H,

have been shown to have a protective effect against any damage that might be caused. Fridovich^® suggested that this is because of a reaction of *0 2' and H2O2

to form the hydroxyl radical. This reaction is known as the Harber - Weiss reaction (first postulated by these workers in 1934^®).

H2O2 + *0 2' O2 + OH" + »0H

The rate constant for this reaction is, however, extremely small (near zero) in the absence of transition metal ions and this has led to the suggestion that these metal ions play an important part in the generation of the hydroxyl radical. The "iron catalysed Harber Weiss reaction" may be represented by the net result of the two

reactions shown below.

Fe^^ - complex + «Og" -> Fe^'" - complex + Og

Fe^'' - complex + HgOg •OH + OH* + Fe^" - complex

The second of these reactions is, of course, a Fenton reaction. Similar reactions may be written for the reaction of copper (If) salts with the superoxide anion. The role of the superoxide ion in reducing iron (III) and copper (II) is not without contention and it has been argued that biological systems are full of reducing agents (eg ascorbate, GSH, NADH, NADPH, cysteine) which could equally well reduce Fe^* to Fe^^ or Cu^'" to Cu*. It seems lil<ely that in vivo the most important of these is ascorbate. The observation that *0H generation can be inhibited by superoxide dismutase in many biological systems is evidence in favour of a role for Harber - Weiss type reactions in vivo.

This propensity of relatively unreactive peroxide ions to form the highly reactive hydroxyl anion in the presence of transition metal ions may explain why in health these metals exist only in tightly protein bound forms in living organisms. Conditions in which the carrier proteins for these molecules are saturated with metal ions, as may be seen with iron in kwashiorkor, or disruption of tissues, as will accompany cell death, can lead to a decompartmentalisation of these metal ions and could lead to an increased flux of free radicals.

The hydroxyl radicals formed as a result of these reactions react with extremely high rate constants with almost every type of molecule found in living cells. It has been calculated, for example, that the *0H radical will have a half life in biological systems of only a few microseconds^^: In consequence the radius of diffusion of the radical from its site of production is estimated to be less than lOOnm. Free radicals of lesser reactivity (for example the superoxide radical or organic products of hydrogen abstraction) and hydrogen peroxide will be able to diffuse further within the cell before reacting or undergoing the Harber Weiss reaction and hence an oxidising effect more distant from their initial site of production.