Structures of imidazolone and oxazolone and the hydrogen bonding between guanine and imidazolone. Protein oxidation is also induced by 1 O 2. The following amino acids, tryptophan, tyrosine, cysteine, histidine, and methionine, can be oxidized by 1 O 2 [ ].
In the case of tryptophan oxidation by 1 O 2 , N -formylkynurenine Figure 14 is a major oxidized product [ , ]. The reported reaction rate coefficient between tryptophan and 1 O 2 is 3. Oxidation of tryptophan residue in a certain protein can be examined with a fluorometer [ ]. For example, human serum albumin HSA has one tryptophan residue, and the intrinsic fluorescence of tryptophan at around nm can be diminished by the oxidative damage. Furthermore, sodium azide NaN 3 , a strong physical quencher of 1 O 2 [ ], effectively suppresses this HSA damage.
From the analysis of the effect of NaN 3 on the HSA damage, the contribution of 1 O 2 -mediated oxidation to the total quantum yield of protein damage can be determined [ ]. Photosensitized 1 O 2 production by porphyrin phosphorus V complexes induces the damage of tyrosinase, which is an enzyme to catalyze the hydroxylation of tyrosine, resulting in the deactivation of tyrosinase [ ].
Oxidation of the amino acid residue by 1 O 2 can cause the deactivation of protein function. Structures of tryptophan and N -formylkynurenine, an oxidized product of tryptophan by 1 O 2.
Example of P V porphyrin photosensitizer. Photocatalyzed 1 O 2 production by TiO 2 may not play an important role in the oxidation reaction [ 31 , 94 ]. Formed 1 O 2 on the TiO 2 surface is quenched by TiO 2 itself with relatively large quenching rate coefficient e. In the presence of bovine serum albumin, 1 O 2 produced by TiO 2 photocatalysis is effectively quenched, suggesting the protein oxidation [ 94 ].
However, in the case of TiO 2 photocatalyst, other ROS are more important for protein oxidation than 1 O 2 -mediated reaction [ 29 , 30 , 31 , 32 ]. ROS detection is an important theme to investigate a biological effect of ROS or evaluation of the activity of PDT photosensitizers [ , , , ].
Fluorometry is one of the most important and effective methods of ROS detection. For example, 5-carboxyfluorescein-based probe has been developed Figure 16 [ ]. This probe can detect H 2 O 2 in the living cell. As an inexpensive method, the fluorometry using folic acid Figure 17 was reported [ 23 , , ]. Using folic acid or its analogue, 1 O 2 can be also detected [ ].
Specifically, in D 2 O, folic acid or methotrexate Figure 17 , an analogue of folic acid, is effectively decomposed by 1 O 2 , resulting in the fluorescence enhancement [ ]. Structure of 5-carboxyfluorescein-based fluorescence probe for H 2 O 2 [ ]. Structures of folic acid and methotrexate and the fluorometry of ROS [ , ]. The pH-dependent control [ ] and target-selective control [ , , , , ] methods have been reported. It has been reported that free base porphyrins were synthesized to control their photosensitized 1 O 2 generating activity by pH Figure 18 [ ].
The S 1 state of this porphyrin is quenched by the electron-donating moiety in neutral or alkali solution. However, protonation of this electron-donating moiety under acidic condition suppresses the electron transfer, leading to the recovery of the 1 O 2 production activity of porphyrin ring.
Because cancer cell is slightly a more acidic condition compared with normal cells [ , , ], this pH-based control of photosensitized 1 O 2 production can be applied to cancer-selective PDT. DNA-targeting control of photosensitized 1 O 2 generation has been also reported [ , ]. For example, electron donor-connecting porphyrins have been studied Figure 19 [ 81 , , , ].
These compounds can be photoexcited by visible light irradiation, and their S 1 states are effectively quenched through intramolecular electron transfer. The charge-transfer state energy can be raised through the binding interaction with DNA, an anionic polymer, resulting in the inhibition of the intramolecular electron transfer and enhancement of 1 O 2 generation.
Example of the reported pH-responsive porphyrin [ ]. Hydrogen peroxide is easily produced from the oxidation processes of chemical compounds by oxygen molecules. Formed H 2 O 2 in cells can be incorporated into cell nucleus and activated by endogenous metal ions. Base modifications lead to carcinogenesis or lethal effect. Photoirradiation to various sensitizing materials induces 1 O 2 production.
Visible light has sufficient energy to produce 1 O 2. Therefore, 1 O 2 is easily produced by various dyes under photoirradiation. Formed 1 O 2 can oxidize guanine residues of DNA without sequence specificity and several amino acid residues of protein within its lifetime, which depends on the surroundings.
Various detection methods of these ROS have been developed. The well-established roles they have in the phagosome and genomic instability has led to the characterisation of these molecules as non-specific agents of destruction. Interestingly, there is a growing body of literature suggesting a less sinister role for this Jekyll and Hyde molecule. It is now evident that at lower physiological levels, H 2 O 2 can act as a classical intracellular signalling molecule regulating kinase-driven pathways.
The newly discovered biological functions attributed to ROS include proliferation, migration, anoikis, survival and autophagy. Furthermore, recent advances in detection and quantification of ROS-family members have revealed that the diverse functions of ROS can be determined by the subcellular source, location and duration of these molecules within the cell.
In light of this confounding paradox, we will examine the factors and circumstances that determine whether H 2 O 2 acts in a pro-survival or deleterious manner. Reactive oxygen species ROS are a family of molecules that include highly reactive free oxygen radicals e. H 2 O 2 and superoxide have been the main investigative foci of ROS biology in recent years, and given the fact that superoxide is rapidly converted to H 2 O 2 in the cell, we will concentrate on H 2 O 2 as the principal ROS member.
Commoner et al. Nearly a decade later, landmark discoveries occurred, identifying two major sources of such ROS, mitochondrial superoxide generation and the phagocytic respiratory burst. The first record of mitochondrial superoxide generation was reported by Boveris et al. In the s, a small collection of studies reported that exogenously added H 2 O 2 could mimic the signalling activity of insulin. It was demonstrated that Nox enzymes have a fundamental role in numerous physiological processes, including survival signalling.
Pivotal to this expanding field of research was elucidating the precise mechanism through which H 2 O 2 and other ROS could modulate signalling pathways. The widely accepted hypothesis proposed by the Tonks group describes reversible inhibition of phosphatases that negatively regulate signalling cascades through oxidation of redox-sensitive cysteine residues.
This review will examine factors such as source and site of H 2 O 2 formation to further discuss this conflicting role of cellular ROS. Notably, they fail to show the diverse physiological functions attributed to the Nox family, and hence, will not be discussed further. Six transmembrane domains form a channel to allow successive transfer of electrons.
This complex comprises a catalytic subunit, the integral membrane protein gp91phox and a p22phox subunit. Activation of this catalytic core relies on the recruitment of several cytosolic protein subunits. Cytokines and growth factors induce ROS production through activation of locally recruited Noxs in non-phagocytic cell types. Nox2 structure. Exposure to pathogenic organism triggers Nox2 complex assembly in the neutrophil by recruiting various subunits to the plasma membrane.
The activated Nox complex then releases superoxide in micromolar concentrations into the phagosome, thus killing the pathogen. Accurate quantification and localisation of H 2 O 2 have been the rate-limiting factors in ROS cell signalling research. This stumbling block is further compounded by recent evidence suggesting that H 2 O 2 is not as freely diffusible as once thought. Miller et al. Subcellular localisation of Nox-generated H 2 O 2. The fluorescence observed shows a staining pattern consistent with the endoplasmic reticulum.
We now understand that non-phagocytic Nox enzymes are no longer confined to the plasma membrane. They have been identified in numerous subcellular compartments such as the endoplasmic reticulum, nucleus and mitochondria.
Ushio-Fukai 29 published a thorough review on the compartmentalisation of Noxs affecting immune signalling pathways. Several papers describing compartmentalised H 2 O 2 survival signalling have emerged recently. A complex array of enzymatic i. Effective antioxidant activity is necessary to buffer fluctuations in the cellular redox status and avoid irreversible oxidation of integral cellular macromolecules such as proteins, lipids and DNA.
Recent data have shown that certain antioxidant members are precisely regulated by Nox-driven signalling to channel H 2 O 2 to colocalised target proteins. Toledano et al. In consequence to growth factor and cytokine stimulation, kinase-driven pathways are thought to both activate Nox activity and moreover, phosphorylate and thus inactivate local Prx1.
This process ensures direct, efficient H 2 O 2 delivery to the target protein. This strangely familiar dichotomy, mirroring ROS biology, is governed by similar parameters, including subcellular location and concentration. The double-edged sword nature of NO reactions is exemplified in the setting of apoptosis. Overproduction of RNS nitrosative stress , as with oxidative stress, can potentially trigger cell death processes such as DNA fragmentation and lipid oxidation.
Understanding the mechanism through which H 2 O 2 modulates signalling pathways is paramount to unearthing the signalling role of Noxs. In keeping with Nox activity, finely controlled, modest fluctuations of the cellular redox status have been shown to be capable of reversible modulation of signalling cascades. Three principal mechanisms of survival pathway activation have been proposed: a inhibition of phosphatases, b activation of tyrosine kinases and c transcription factor activation Figure 3.
Activation of Nox activity occurs upon growth factor stimulation. This happens through recruitment of various protein subunits or by induction of Nox isoform expression. The cysteine residues located at the active sites of specific phosphatases are susceptible to reversible oxidation. This oxidation results in the inhibition of these phosphatases that negatively regulate survival signalling, thus propagating a pro-survival effect.
Nox-generated ROS can also stimulate many pro-survival kinases Src and transcription factors NF- k B , resulting in enhanced survival signalling. A large body of evidence identifies cysteine residues as the most likely targets of Nox-generated ROS. Reversible oxidation of cysteine residues occurs when sulphenic acid intermediates Cys-SOH are formed. Reversal of this reaction is mediated by incubation with thiol compounds.
Interestingly, this process of protein reduction is thought to be equally significant as Nox enzymes in the redox regulation of signalling pathways. Phosphatases, a structurally diverse family of receptor-like non-transmembrane enzymes, target specific substrates in vivo and are critical regulators of signalling pathways.
The presence of a conserved arginine residue confers an unusually low p K a , hence rendering the cysteine residue highly susceptible to oxidation. Colocalisation of Nox4 and PTP-1B at the endoplasmic reticulum results in enhanced extracellular signal-regulated ERK signalling and proliferation upon reversible cysteine oxidation. For example, upon cell attachment to the extracellular matrix ECM and associated generation of H 2 O 2 , the tyrosine kinase Src becomes oxidised at two cysteine residues and thus becomes activated.
H 2 O 2 can modulate enzyme activity by several differing mechanisms Figure 3. Identification of specific protein targets of Nox-mediated ROS is vital to delineating their ever-expanding roles in cellular signalling pathways. Nox activation in non-phagocytic cell types varies considerably and continuingly drifts away from the prototypical Nox2 paradigm. Numerous stimuli increase non-phagocytic Nox expression. Given the large amount of contrasting data, induced expression of particular Noxs appears to be both stimulus and cell type-specific.
Duox1 and Duox2 are widely and differentially expressed throughout most human tissues. Induction of Duox1 and Duox2 expression has been elicited in response to Th1 and Th2 dominant cytokines, respectively.
Expression of Nox3 and Nox5 has been documented in various human tissues; however, characterisation of specific promoter regions and transcription factors remains unchecked. Upregulation of Nox activity accounts for growth factor-induced ROS production in most cases of Nox pro-survival signalling. Nox1 and Nox3 tend to follow the original Nox2 model such that a stimulus triggers the formation of the active Nox complex, coupled with various combinations of protein subunits.
Since the double bonds are often what causes the molecules to absorb light, and therefore give the molecule its colour, removal of them destroys the pigments and so removes the colour. H 2 O 2 is used in this way to bleach wood pulp to make white paper, and the melanin in hair. Some of the most famous Hollywood movie stars, such as Marilyn Monroe and Jean Harlow, were 'peroxide blondes' and perhaps owe some of their fame to the H 2 O 2 molecule. One advantage of hydrogen peroxide over some other bleaching agents, such as chlorine gas, is that the decomposition products, water and oxygen, are not harmful.
Hydrogen peroxide also plays a less beneficial role in modern life, as one of the causes of photochemical smog. They can aim this spray accurately in any direction. Usually in the wild they are aiming at predators like ants.
The bombardier beetle stores a mixture of the hydrogen peroxide and hydroquinones. At the moment that the defence is needed, the mixture is pumped into a reaction chamber, where it is mixed with catalysts catalases and peroxidases which catalyse the reaction decomposing the peroxide to oxygen. This oxidises the hydroquinones to quinones, very exothermically. The oxygen gas propels the hot spray out of the jet on the abdomen. Well, first there was the Messerschmitt rocket aircraft "Komet". This wasn't a conventional jet aircraft, it was actually the only rocket aircraft to ever fly in operational service in WW2.
It was fitted with a rocket motor that made it far faster than any other fighter in the air, but the fuel used made it somewhat "unsafe". It is said that any organic matter including humans could spontaneously combust in contact with the T Stoff.
The fuels were loaded separately, with the aircraft and crew washed down carefully after the first fuelling, and again after the second one. The explosive reaction produced a mixture of hot gases, steam and nitrogen:. The plane would be doing mph by the edge of the airfield, climb vertically at mph, then approach American bombers at over mph.
No Allied fighter could keep pace with it, but the Me's of JG only shot down 9 bombers, and lost far more of their own in accidents.
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