Free Radical Damages In Proteins

by Adomas Urbanavicius


The Principles Of Free Radical Chemistry
Free Radicals Damages On Amino Acids And Proteins

1. Introduction

Free radicals are molecular species which contain an unpaired electron (usually represented as R·). Consequently, they are some of the most chemically reactive molecules known. Because of the need to pair its single electron, a free radical must abstract a second electron from a neighbouring molecule. This causes the formation of yet another free radical and self-propagating chain reaction ensues.

Free radicals in human body can arise from fatty food, smoking, alcohol, environmental pollutants, hydrogen peroxide, pollutants, ozone, toxins, carcinogen toxins, ionisation etc. The vast majority of free radicals come from within the body, an unavoidable by-product of living system. Free radical intermediates are produced in living systems under normal conditions, the body handles free radicals formed by the breakdown of compounds through the process of metabolism. The major sources of free radicals (such as O2- and HO2·) are modest leakages from the electron transport chains of mitochondria, chloroplasts and endoplasmic reticulum.

The resulting free radicals, such as superoxide anion (O2-) and hydroxyl radical (OH·), as well as the non-radical hydrogen peroxide, can damage macromolecules, including DNA, proteins and lipids. Likewise, other products of oxygen metabolism, such as hypochlorous acid, chloramines, and oxidised lipids have all been related in such damages. The superoxide radical, although it is unreactive in comparison with many other radicals, biological systems can convert it into other more reactive species, such as peroxyl (ROO·), alkoxyl (RO·) and hydroxyl (HO·) radicals.

There are four types of free radicals damages:

  1. Damage to fat compounds: The fatty membranes surrounding the cells being the prime target to free radicals attacks. The damaged membranes then loose its ability to transport oxygen, nutrients or water to the cells.
  2. Damage to protein molecules: Free radicals also attack the nucleic acid which comprise the genetic code within each cell. The nucleic acids function is to regulate the normal cell function, growth and also to repair the damaged tissues.
  3. Cell damage: Damages done to the chromosoins and nucleic acids might initiate the growth of abnormal cells, which is the first step in cancer development.
  4. Lysosomes damages: Lysosomes are little sacs in the cell that contain degenerative enzymes. The enzymes leak out when the membrane cell breaks and they start digesting the cell itself, spreading to nearby cell causing a chain reaction of destruction which, eventually, will lower the immune system resistance.
And such production of reactive oxygen species and other free radicals and theirs damages to various molecules and cells may result not only in the toxicity of xenobiotics but also in the pathophysiology of ageing, and various age-related diseases, including cataracts, arteriosclerosis, neoplastic diseases, diabetes, chronic inflammatory diseases, cancer and etc.,

Fortunately, the body also has several natural chemical means or systems for neutralizing free radicals. There is agents that counteract and minimize free radical damage and their function is to donate or provide unpaired electrons to which free radicals can attach without causing harm. Such "cell-savers" are called "Antioxidants."

Antioxidants get their name because they combat oxidation. Oxidation is a reaction in which a molecule looses an electron. The two major sources of antioxidants are:

  1. Those that you get from food or food supplements
  2. Those produced within your own body.

And there are some types of antioxidants, like the Flavonoids, found in the skin and seeds of fruits, possess the ability to physically capture free radicals until these are actually removed from the body. Others, like Sulphorophane, found in broccoli, tend to enhance the body’s own free radical scavenging mechanism. And finally, the ones like L. Limonene, phytochemical found in citrus fruit peels, can actually perform both actions. Some popular antioxidants today include Vitamin E, Vitamin C, Vitamin A, which can be taken under a health supplement form or through fruits, vegetables, fish oil, green tea, sesame oil, and Genistein from soy bean shown to be cancer preventive. Some antioxidants come from minerals, such as selenium, copper, zinc (they are considered antioxidants because they work together in conjunction with an antioxidant enzyme and are necessary for the enzyme to function properly).. etc. The list of dietary antioxidants goes on and on, and scientist are continually discovering more.

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2. The Principles Of Free Radical Chemistry

In order to appreciate the role of free radicals in biology it is firstly necessary to understand their chemical nature. In general, molecular bond formation is dependent on the interaction of paired electrons. They have properties which enable them to spin about their own axes, and to confer bond stability they must be paired in opposite spins. Free radicals and molecular oxygen have properties which do not conform with their normal requirements of bonding. When a conventional molecular bond is broken, the two composite electrons usually split heterolytically:

However, under certain circumstances it is also possible for bonds to break homolytically:

This is a free radical reaction. In non-biological systems, free radicals can be produced via the effects of ionising radiation, temperature, and various photochemical events.

Often, free radical reaction involve, either directly or indirectly, the formation of oxyradicals. Molecular oxygen is, in fact, a biradical possessing two unpaired electrons of parallel spin (Fig.1). This electrons exist at their lowest energy level when unpaired and when they have spins in the same direction. This configuration is called the ground or triplet state and it describes the paramagnetic and electronic behaviour of oxygen.

Most molecules have electrons with opposite spins and are termed diamagnetic, their ground states are described as singlets. Two forms of singlet oxygen exist (Fig.1).

The transfer of electrons to oxygen can also lead to the production of toxic free radical species. The best documented of these is the superoxide radical, shown in Fig.1. A two electron reduction of oxygen results in the formation of the peroxide ion (Fig.1). This is not a free radical, but is very reactive and at physiological pH protonates to form hydrogen peroxyde (H2O2). The reaction of hydrogen peroxyde with superoxide radical can lead to a series of reactions wich produce additional reactive species. These are described below.

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2.1. Haber - Welss and Fenton reactions

Haber proposed a reaction between superoxide radical and hydrogen peroxide

The product is the hydroxyl free radical (OH·), reputed to be one of the most reactive molecules known in chemistry. This original reaction was later found to occur in two steps, now called "Fenton reaction". Firstly, the superoxide radical reduces the ferric ion:

Secondly, this interacts with H2O2 to produce the OH· radical:

The total sequence of events can be resumed as:

There is still some controversy over the Fenton reaction particularly in vivo, and it is thought that OH· may not be the only oxidising radical found in systems containing Fe3+/2+, O·2- and H2O2. The high reactivity of these other species makes them very difficult to study and they have been aptly named the "crypto OH·" radicals. When protonated in aqueous solutions, O·2- forms the reactive molecule, HO·2. It is thought that this species is also produced in vivo.

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2.2. Termination and propagation reactions

The fate of the free radicals is an important consideration when evaluating their toxicity. Because of the intrinsic need for their electrons to pair, they are highly reactive and has very short half life (OH· has life of 10-9 seconds). Of course, some radicals such as semiquinone radical present in tobacco smoke has a half life of several days. The termination reactions of the free radical reactions have been categorised by Slater into several types:

Free radicals often reacts with other molecules in a manner that initiates the formation of many more free radical species. It is this self-propagating ability that makes them so toxic to living organisms. Thus, free radicals are generated by several mechanisms:

A single initiation event which lead to the production of one free radical can soon produce many more; their is called "cascade", and it can be prevented via the termination steps above. In cells, termination also occurs by the interaction of protective mechanism and antioxidants.

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2.3. Singlet oxygen

However, as stated earlier a second and most important reaction of oxygen is the spin reversal of one of the parallel electrons in the outer orbital. This new arrangement of the molecule is called the singlet state, and hence the term singlet oxygen (1O2). Two singlet states exist. The first type of singlet oxygen (Fig.1, where g represents a constant defining the energy value of a molecule in a magnetic field) is most important in vivo. The second is energetically unstable and usually decays to first state before it has time to react with other molecules. It is important, of course, to note that (1O2) is not a free radical, but because of its electronic arrangement it displays properties which are similar to the free radical species of molecular oxygen. And there are some important non-photochemical reactions with this type of oxygen.

Photosensitised reactions are very important too, because they are involved in production of cellular (1O2) . A photosensitive molecule is one which on activation by radiation or light causes another molecular component to react. Two types of photoreactions exist:

A sequence of events is initiated by the absorption of a photon by the sensitive molecule (by carotenoid or chlorophyll), and can be summarised into the Type I and Type II reactions as follows:

The flux along these pathways is very much dependent on the concentrations of the substrate and molecular oxygen. If the concentration of oxygen is high but the substrate concentration is low, the Type II reactions is favoured. Type I reaction are more efficient if the reverse conditions are prevalent. The fate of the excited species in both types of reaction is most important, particularly as more excited species and free radicals can be produced. Sensitises can also undergo electron transfer reactions with the substrate or oxygen:

Formed (1O2) and various types of free radicals can react with many different organic molecules especially lipids. As a consequence, membranes are major targets of attack. The nucleic acids (particularly guanine), proteins and enzymes, are also susceptible. And in next section we will try to describe free radicals damages to proteins.

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3. Free Radicals Damages On Amino Acids And Proteins

3.1. Review

Radical-mediated protein oxidation has been studied throughout the century. In the first decade , Dakin published detailed chemical studies of the oxidation of leucine and other amino acids by Fenton systems (transition-metal ion plus hydrogen peroxide), and protein agregation and fragmentation was detected by others. Soon after the discovery of gluthatione, Hopkins appreciated that this reductant could be both an anti- and pre-oxidant and inactivate proteins. Several authors assessed the proteolytic susceptibility of oxidized proteins, and demonstrated biphasic effects, whereby limited oxidation leads to enhanced susceptibility, while more extensive oxidation may be associated with increasing resistance.

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3.2. Protein oxidation chemistry

In many works there was detected that amino acids in many various reactions can form carbonyls, such as the oxo acids and aldehydes with the same or less carbon atom then the parent amino acid; e.g. glycine giving rise to glyoxsal and glyoxylic acid, formaldehyde and formic acid; alanine giving rise to acetaldehyde and acetic acid, etc. This general scheme has been confirmed for many amino acids, including aromatic amino acids, although other reactions may also occur.

During the oxidation of aliphatic amino acids by HO·, hydroxilated derivates, notably of the side chains, are formed. During the oxidation of aromatic residues, the formation of phenoxyl radicals from tyrosine, and their conversion into dityrosine and further products, can occur, especially if there are no reductants to repair the tyrosil radicals (e.g. thiols, vitamin E) and if there are vicinal tyrosil radicals. Hydroxylation of phenylalanine, tyrosine and tryptophan is also a characteristic reaction of hydroxyl radicals, and similar reactions of histidine (giving 2-oxohistidine) are important. Histidine in reactions with free radicals can form some imidazole decay products or in some cases aspartic acid and can form some histidine derivates (Fig.2.). Fenton chemistry can generate both the aliphatic and aromatic products.

Early radiation studies on lysozyme, ribonuclease and other enzymes were carried out mainly in the absence of O2 and showed that HO· was the most effective inactivator, and characterised other more selective (but less efficiently inactivating) species such as (SCN)2-·, Br2-·, Cl2-· and I2-·. For example, (SCN)2-· was found to react with an important tryptophan residue in pepsin and so inactivate the enzyme, although damage could be reversed.

Table 1. summarises some products of protein oxidation. Schemes 1 and 2 illustrate some of the major reactions believed to be important in the oxidation of the side chain and the backbone respectively. Alkoxyl radicals apparently have a greater importance in protein oxidation chains than they do in lipid peroxidation, in which peroxyl radicals are key chain propagating species.

TABLE 1. Some product of protein oxidation
Oxidative insult
Tyr+HO· or RNIs (reactive nitrogen species) Dopa
Tyr+HOCl 3-Chlortyrosine
Tyr+RNIs 3-Nytrotyrosine
Tyr+HO·, or one electron oxidation of Tyr or HOCl, followed by radical- radical combination Dytirosine
Phe+ HO·, one electron oxidation o- and m-tyrosine
Phe+ HO·, before or after dimerization Dimers of hydroxylated amino acids
Trp+ HO·,or on electron oxidation N-Formylkynurenine; kynurenine
Trp+ HO·,or on electron oxidation 5-Hydroxytryptofan; 7-hydroxytryptofan
His+ HO·, or one electron oxidation 2-Oxohistidine
Glu+ HO· in presence of O2 Glutamic acid hydroperoxide
Leu+ HO· in presence of O2 Leucine hydroperoxides and hydroxides; a-ketoisocapronic acid; isovaleric acid; isovaleraldehyde; isovaleraldehyde oxime; carbonyl compounds;
Val+ HO· in presence of O2 Valine hydroperoxides and hydroxydes; carbonyl compounds;
Lys+ HO· in presence of O2 Lysine hydroperoxides and hydroxydes; carbonyl compounds;
Pro+ HO· in presence of O2 Proline hydroperoxides and hydroxydes; 5-hydroxy-2-aminovaleric acid; carbonyl compounds;
Arg+ HO· in presence of O2 5-Hydroxy-2-aminovaleric acid;
Ile+ HO· in presence of O2 Isoleucine hydroperoxides; isoleucine hydroxides; carbonyl compounds;
Gly: hydrogen atom abstraction from a-carbon followed by reaction with CO2·- radicals; Aminomalonic acid
Met+ HO· or one electron oxidation Methionine sulphoxide
Cys+ HO· or other hydrogen atom abstracting species Cystine; oxy acids
All amino acids exposed to photo-oxidation, oxidizing radicals or HOCl RCHO species formed by decarboxylation or deamination

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3.3. Protein oxidation by lipid derived species

It is well established that lipid radicals may damage proteins. Such reactions show differences from those observed with radiolytic systems. End-products of lipid peroxidation, such as malondialdehyde and 4-hydroxynonenal are also inactivating agents, possibly via Schiff base formation. Schiff bases are short lived species formed by the reaction of carbonyl groups with amines, and can be formed during exposure of proteins to lipid derivated aldehydes, autoxing sugars and amino acid derivated aldehydes. In systems that contain lipid hydroperoxides and soluble proteins, metal ion catalysed reactions appear to be central. Protein inactivation by peroxidizing lipid is often associated with the binding of lipid components of protein. Thus, in membranes, competition and interactions between protein and lipid oxidation are expected. Metal ion catalysed damage to unirradiated membranes seems to affect lipid and protein in parallel, and is restricted by a-tocopherol. Membranes can also influence radical damages to proteins by physical separation of the target from the radical source or by sequestration of transition metal ions away from hydroperoxides. Lipid oxidation products, notably aldehydes, can modify the lysine residues. During metal ion and radiolytic attack, lipid and protein oxidation are often concurrent, and occur even while much vitamin E remains. In contrast, hypochlorite selectively attacks the protein, consuming mainly lysine, tryptofan, cysteine and methionine residues, and giving rise to chloramines.

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3.4. Summarized scheme of proteins oxidation in vivo

Scheme 3 summarizes the mechanisms by which oxidized proteins may undergo further reactions in vivo.

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4. References

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