Oxidation of Methionine by Singlet Oxygen - Essay Example

?Introduction Oxygen was discovered in 1775 by Joseph Priestley (Priestley, 1775). Later, Avagadro established the diatomic nature of oxygen, and itsparamagnetic properties were investigated by Faraday in 1811 (Parkes, 1967). Spectroscopy was used to prove the existence of higher energy state of oxygen, which later was called singlet oxygen. (Herzberg, 1934). Over the past twenty five years significant increase in data regarding singlet oxygen has led to the recognition of its importance in organic chemistry, biochemistry and medicine. Singlet oxygen is an extremely reactive, nonradical, electrophilic molecule. Its electron arrangement is different from abundant normal diradical triplet oxygen. Singlet oxygen can be formed from triplet oxygen with the assist of photosensitizers, like Rose Bengal. Such compounds use light to convert triplet into singlet oxygen. Due to low activation energy and its electron configuration singlet oxygen is a highly reactive molecule and can readily form bonds with a number of organic molecules. It is less stable than the normal form of oxygen. However, depending on the environment singlet oxygen has the possibility to exist for more than one hour at room temperature. (Schweitzer, et al., 2003) One of the reactions that singlet oxygen can take part in, is the oxidation of amino acid residues of proteins. Cysteine and methionine residues are particularly susceptible to such forms of oxidation. In contrast to oxidation of other amino acids, this type of oxidation is reversible. Both R- and S-stereoisomers of methionine sulfoxide form as a consequence of methionine residue oxidation. The reaction can be reversed with the aid of stereospecific methionine sulfoxide reductases which can be found in most cells. The enzyme catalyses the formation of methionine residues from methionine sulfoxide (Carey, et al., 1984). The fact that the quantity of methionine sulfoxide increases over ages and leads to age related deceases highlight the paramount importance of investigations in this area. For that reason a number of aging models were studied. In all models the importance of methionine sulfoxide reductase (Msr) was established. Mutations leading to decrease in Msr lead to decrease in life span, but the reverse process significantly increases it. (Stadtman, et al., 2005) This work, for example, uses methionine oxidation by singlet oxygen, generated using Rose Bengal and laser as a radiation source. This report is initially focused on describing the key literature associated with oxidation of methionine by singlet oxygen, highlighting relevant for the project issues and arguments, then moving on to presenting the research that has been done so far, making accent on the methods used and results achieved, before finally identifying the existing gaps in the study and setting up a plan for future work. Singlet and triplet oxygen Triplet oxygen is the most stable and abundant form of oxygen. The difference between two forms of oxygen can be seen by comparing the molecular orbitals of both forms (Figure 1). Figure 1: Triplet (left) and singlet (right) form of oxygen. (Min and Lee, 1999) (Frimer, et al., 1985) To define spin states of molecules spin multiplicity is used. It can be described as 2S+1. Here S is the spin quantum number, which is 1 for triplet oxygen. Therefore, the spin multiplicity for triplet oxygen is 3 and it is paramagnetic. Triplet oxygen easily reacts with radicals, but peptides in general and methionine in particular are in the singlet state. Singlet oxygen, in contrast, has different electron configuration in the ?-antibonding orbital. It is a non-radical, electrophilic molecule. The spin quantum number is 0 and multiplicity is 1, these parameters characterise the form of oxygen presented on the Figure 1 as a singlet. It does not follow the Hund’s rule, extremely reactive and 22.5 Kcal/mole higher in energy then triplet oxygen. Five excited states are produced due to electron repulsion. The 1? represents the singlet form which is responsible for the reaction with methionine and oxidation in foods. Temperature has little effect on the reaction rate due to low activation energy. The existence time of singlet oxygen depends on the solvent that surrounds the dissolved molecules. Singlet oxygen reacts directly with double bonds with no free-radical intermediates observed. This reaction is particularly important in case of vitamins and lipids, as they are most susceptible to damage by oxidation. (Korycka-Dahl and Richardson, 1978, Girotti, 1998). Singlet oxygen formation Singlet oxygen can be produced in photochemical, enzymatic and chemical processes (Krinsky, 1977). In photochemical processes photosensitizers absorb energy from light and use it to convert triplet oxygen into its singlet form. Photosensitizers include compounds like myoglobin, riboflavin, chlorophyll, porphyrin. However, in this particular research Rose Bengal is used. Photosensitizers absorbs visible or ultraviolet light which makes it a singlet, unstable molecule (1Sen*). The excited molecule can emit radiation in the form of fluorescence and return to the ground state or, using intersystem crossing form the excited triplet state which is lower in energy (3Sen*). The formed triplet photosensitizer (3Sen*) reacts with triplet oxygen to convert it into the singlet. The photosensitizer also converts to singlet and can continue generation of singlet oxygen (Figure 2) (Kochevar and Redmond, 2000). Figure 2: Singlet oxygen formation in the presence of photosensitizers. (Min and Boff, 2002) Talking about employing this process in the functionalization of various substrates, there are two types of process. In the first type of functionalization the triplet form of photosensitizer reacts with an organic substrate, removing electrons or a hydrogen atom from it. The mechanism produces free radicals and is known as Type I. Formed radicals remove hydrogen from other molecules and continue the chain reaction. These radicals can also react with triplet oxygen to form peroxides. The triplet form of photosensitizer is able to react with triplet oxygen leading to superoxide oxygen anion (Foote, 1976). In contrast to Type I, in Type II mechanism 3Sen* reacts with triplet oxygen. The reaction leads to the formation of singlet oxygen which later reacts with organic substrates forming peroxides. The reaction proceeds almost quantitatively and includes energy transfer from excited photosensitizer to the unexcited form of oxygen. The reaction rate as well as the choice between Type I and Type II mostly depend on the reaction medium (Kepka and Grossweiner, 1972) (Figure 3). Figure 3. Type I and Type II formation of the triplet form of photosensitizer and transformation of organic substrates into peroxides (Min and Boff, 2002). Both types of reactions will increase the oxidation rate by either produce singlet oxygen or form reactive radicals. Organic substrates and triplet oxygen compete for the triplet active photosensitizer and this competition determine whether the reaction is Type I or Type II. Talking about the particular case of Rose Bengal it can be stated that the formation of singlet oxygen proceeds according to the mechanism represented on Figure 4. Figure 4: The use of Rose Bengal as a photosensitizer. Initial absorption of light which transforms Rose Bengal into the singlet state is followed by intersystem crossing with the formation of the triples state of this photosensitizer. This reaction is followed by the formation of singlet oxygen. The produced form of oxygen is reactive enough to oxidase organic substrates. However, this reaction is not the only possible one. Singlet oxygen can either loose energy and return to the triplet form or interact with organic substrate without any chemical reaction but also with the formation of triplet oxygen. The described reactions are described using reaction constants kd, ksa, ksp. Because ? of singlet oxygen in water is 2 ?s (2?10-6 s) kd can be calculated as 1/? which will give 5?105 s-1. For methionine ks=2?107 M-1? s-1and using the formula for kt kt = kd + ks[S] (1) this reaction constant can be calculated for the corresponding concentration of methionine: kt = 5?105 s-1 + 2?107 M-1? s-1 ?10-2 M = 7 ?105 s-1 For a range of different methionine concentrations different kt will be produced. Taking into account the formula (1) the following plot can be constructed. ks represents the slope and for this case is 2?107 M-1? s-1 (Du, et. al., 1998). kt, s-1 [S], M 7 ?105 1?10-2 9 ?105 2?10-2 11 ?105 3?10-2 13 ?105 4?10-2 Figure 5: Linear dependence between concentration [S] and constant kt Attack of singlet oxygen on lipids Formed in the described processes singlet oxygen can attack proteins. These attacks target specific amino acids and lead to fragmentations, modifications and changes in the overall charge of the protein. Each amino acid reacts with singlet oxygen in its own way. The susceptibility of amino acids for oxidation can be altered by changing the primary, secondary or tertiary structure of the peptide. In spite of unique properties of amino acids, it was noted that sulphur containing amino acids are particularly susceptible to oxidation. Hydrogen atom can be removed from cysteine with the formation of thiyl radical. This radical combines with another thiyl radical and forms a disulphide bridge. Oxygen can also attack methionine residue with the formation of methionine sulphoxides. Both reactions are reversible and can be realised using the corresponding enzymes (Figure 6) (Farr and Kogama, 1991). Figure 5: Methionine oxidation by singlet oxygen (Stadtman and Barlett, 1997). Experimental part The experimental work includes two experiments. The first experiment includes the investigation of methionine oxidation under different concentrations. The concentration of Rose Bengal was constant in all experiments. Oxygen uptake was measured using oxygen electrode and for all the experiments the oxidation rate was calculated. The process is followed by Michaelis-Menten kinetics, for this reason the rate is can be salculated using the equation bellow: v0= (2) here: v0 is the initial rate of the studied reaction; Vmax is the maximum rate of the same reaction. If the amount of methionine is increased Vmax will also increase due to the following relationship: Vmax = kcat[methionine] Km- Michaelis constant; [S] – methionine concentration; If assumed that only one product is formed in the oxidation reaction then the process represented on Figure 4 should be changed: Figure 5: Oxidation of methionine leading to only one type of products. For the collection of the required amount of information for the described equations the six different methionine water solutions were prepared. The volume was constantly 3 ml and 40 ?L of Rose Bengal were added in every case (Table 1). № V(H2O), ml V(Methionine), ml V(Rose Bengal), ?l [Methionine], mM 1 2.5 0.5 40 ?l 8.33 2 2.0 1.0 17.67 3 1.5 1.5 25.00 4 1.0 2.0 33.67 5 0.5 2.5 42.00 6 0.0 3.0 50.34 Table 1: Preparation of different concentrations of methionine. The oxygen consumption was measured using an oxygen electrode. The maximum consumption rate was subsequently found to be 240 ?mol/litre/minute. Schematically, the oxygen consumption resembles the patterns represented on the Figure 6. Figure 6: Schematic representation of the oxygen consumption patterns at different methionine concentrations. If R is determined for several substrate concentrations it is possible to determine Rmax and Km. Because the process is followed by Michaelis-Menten kinetics, the dependence between methionine concentration and the maximum rate of oxygen uptake should became of a rectangular hyperbola. Figure 7: Schematic representation of Michaelis-Menten kinetics during oxygen uptake. On the figure 7 (R/[Met]), with the increase in the concentration of methionine the rate approaches the maximum reaction rate. Here Michaelis constant is equal to substrate concentration at which R=1/2 Rmax. Therefore, Rmax and Km can be found from the Michaelis-Menten plot. If this method is employed only approximate values for Rmax and Km can be determined. To achieve exact results a Lineweaver-Burke plot must be constructed (1/R versus 1/[S]) (Mathonet, et al., 2006). By using the equation for Michaelis-Menten kinetics and taking its reciprocals the following equation will be produced: Also, the following calculations are true for the particular case of this experiment: represent the slope The equation is used to construct the Lineweaver-Burke plot (Figure 8). Figure 8: Schematic representation of the Lineweaver-Burke plot. By extrapolating the line to the Y, the exact value for the reaction rate can be sound. (230 ?mol/litre/minute) Talking about finding the exact concentration of the Rose Bengal, Beer-Lambert equation was used. A= ??c?l For this reason, c = A/(??l) here A = absorbance measured. In this case it was measured to be 1. ? = extinction coefficient, 105 M-1 cm-1, l = the cuvette thickness, cm, c = concentration of Rose Bengal, M c = 1/(105 ? 1) = 10-5 M A similar approach was used in the second experiment. In this experiment also oxygen consumption by methionine was measured but the active singlet oxygen is generated by using laser pulse. The process can be represented on the Figure 9. Singlet oxygen can transform into its triplet form by intersystem crossing or react with the substrate (T) to generate products and triplet oxygen. The reaction with the substrate corresponds to two reactions that are combined on figure 9 and represented as one process. However, the process can be represented in more detail (Figure 10)(Wilkinson, et al., 1995). Figure 9: Using laser pulse in generation of active singlet oxygen and subsequent oxidation of the corresponding substrate. Figure 10: Generation of singlet oxygen and its interactions with the substrate. In case of conducting the reaction in water ?1O2 was determined to be 2 ?s with ks=5?105 s-1. kobs was found to be 108 M-1 s-1 with the concentration of methionine 10-2 M. Consequemtly, K (Figure 10) was calculated to be 106 s-1. In deuterated water was much larger (60 ?s). But methanol was in the middle between normal and deuterated water (12 ?s). In this case also oxygen electrode was used to monitor the reaction. The process can be schematically represented on the following figure: Figure 11: Schematic representation of oxygen consumption using laser. Reaction with methionine can be described on the figure bellow: Figure 12: Reaction of methionine with singlet oxygen. To conclude it can be stated that using Rosa Bengal it was possible to successfully study the kinetics of methionine oxidation. In future it is important to investigate the quantitative methionine oxide detection methods. Study the possible applications of chromatography for this reaction. Figure 13: Schematic representation of the oxygen consumption patterns at different methionine concentrations.(Graphs represented on one plot on figure 6) Figure 14: Schematic representation of oxygen consumption using laser. 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