Taurine Bromamine: Reactivity of an Endogenous and Exogenous Anti-Inflammatory and Antimicrobial Amino Acid Derivative

Taurine bromamine (Tau-NHBr) is produced by the reaction between hypobromous acid (HOBr) and the amino acid taurine. There are increasing number of applications of Tau-NHBr as an anti-inflammatory and microbicidal drug for topical usage. Here, we performed a comprehensive study of the chemical reactivity of Tau-NHBr with endogenous and non-endogenous compounds. Tau-NHBr reactivity was compared with HOBr, hypochlorous acid (HOCl) and taurine chloramine (Tau-NHCl). The second-order rate constants (k2) for the reactions between Tau-NHBr and tryptophan (7.7 × 102 M−1s−1), melatonin (7.3 × 103 M−1s−1), serotonin (2.9 × 103 M−1s−1), dansylglycine (9.5 × 101 M−1s−1), tetramethylbenzidine (6.4 × 102 M−1s−1) and H2O2 (3.9 × M−1s−1) were obtained. Tau-NHBr demonstrated the following selectivity regarding its reactivity with free amino acids: tryptophan > cysteine ~ methionine > tyrosine. The reactivity of Tau-NHBr was strongly affected by the pH of the medium (for instance with dansylglycine: pH 5.0, 1.1 × 104 M−1s−1, pH 7.0, 9.5 × 10 M−1s−1 and pH 9.0, 1.7 × 10 M−1s−1), a property that is related to the formation of the dibromamine form at acidic pH (Tau-NBr2). The formation of singlet oxygen was observed in the reaction between Tau-NHBr and H2O2. Tau-NHBr was also able to react with linoleic acid, but with low efficiency compared with HOBr and HOCl. Compared with HOBr, Tau-NHBr was not able to react with nucleosides. In conclusion, the following reactivity sequence was established: HOBr > HOCl > Tau-NHBr > Tau-NHCl. These findings can be very helpful for researchers interested in biological applications of taurine haloamines.


Introduction
Hypochlorous acid (HOCl) and hypobromous acid (HOBr) are microbicidal agents produced when the white blood cells, neutrophils and eosinophils, respectively, are challenged by stimuli like bacteria and fungi [1]. Eosinophils, in particular, are associated with the host defense against parasitic helminth infections and asthma exacerbation [2,3]. These halogenating and oxidizing agents also play an important role in tissue damage associated with chronic inflammatory diseases [4,5]. For instance, eosinophilia, a characteristic of asthmatic subjects [6] has been associated with an increased level of 3-bromotyrosine, a biomarker of the damaging effects of HOBr [7].
The formation of these oxidants is due to the large amount of the enzymes myeloperoxidase (MPO) and eosinophil peroxidase (EPO) in these cells, which promote the catalytic oxidation of chloride (Cl´) and bromide (Br´) to HOCl and HOBr, respectively [8,9]. The catalytic mechanism involves the

Preparation and Stability of Tau-NHBr
Tau-NHBr can be prepared by reacting HOBr with taurine (Equation 1). However, it exists in equilibrium with its dibromamine form (Tau-NBr 2 ) and, as has been demonstrated by Thomas et al., pure Tau-NHBr is only obtained using a large excess of taurine. In our studies, we usually prepare stock solutions of Tau-NHBr by reacting 5 mM HOBr with 500 mM taurine in 50 mM phosphate buffer, pH 7.0, i.e., a 100-fold excess of taurine. This solution was very stable when stored in the refrigerator and lost less than 20% of its initial concentration after five weeks [33].

Reactivity with Tryptophan
Aiming to obtain a comprehensive knowledge of the chemical reactivity of Tau-NHBr, we used endogenous and non-endogenous compounds as potential targets. The first target was tryptophan, whose reactivity with HOCl and HOBr has been described [34,35]. Figure 1a shows the time-dependent fluorescence decay of tryptophan by the addition of Tau-NHBr. In these experiments, the concentration of tryptophan was kept constant at 25 µM and the concentration of Tau-NHBr was varied from 100 to 500 µM. From this pseudo-first-order experimental condition, the apparent second-order rate constant (k 2 ) was calculated (7.7ˆ10 2 M´1s´1, Figure 1b). To gain insight into the significance of this value, it was compared with those obtained using HOBr, HOCl and Tau-NHCl. Figure 1a shows that, whereas 150 µM Tau-NHBr provoked the tryptophan fluorescence decay in about 20 s, HOBr caused the same effect in less than 0.02 s (Figure 1c). Unfortunately, this reaction was too fast to determine k 2 ; however, its higher reactivity compared to Tau-NHBr was evident. Regarding HOCl, we took as reference a recent determination performed in our laboratory using exactly the same analytical protocol (k 2 = 8.1ˆ10 4 M´1s´1) [15]. Finally, and in agreement with the well-known low reactivity of Tau-NHCl [31], we found that this haloamine was unreactive with tryptophan. In fact, the experiments were done using up to a 10-fold excess of Tau-NHCl compared with Tau-NHBr, but no indication of consumption of tryptophan was observed for up to 10 min. From these results, the following reactivity sequence was obtained: HOBr > HOCl > Tau-NHBr > Tau-NHCl (unreactive).
Besides tryptophan, the reactivity of Tau-NHBr with melatonin and serotonin was also obtained. These tryptophan derivatives have many physiological functions, including endogenous antioxidative activity [36,37]. Among others factors, this property is related to the lower one-electron reduction potential of these molecules compared to tryptophan (tryptophan E˝' = 1.01 V, melatonin E˝' = 0.95 V, and serotonin E˝' = 0.65 V) [38]. In agreement, the k 2 values obtained for melatonin (7.3ˆ10 3 M´1s´1) and serotonin (2.9ˆ10 3 M´1s´1) were about 10-fold higher compared to tryptophan ( Figure 2).

Reactivity with Dansylglycine
In contrast to tryptophan, which has significant intrinsic fluorescence, other oxidizable amino acids, like methionine and cysteine, are not fluorescent. Tyrosine is also fluorescent, but with its maximum excitation/emission at 280/305 nm, our application of a stopped-flow system coupled to a LED source (280 nm) and the emission cut-off filters (305 nm) made the determination of k 2 unworkable. Thus, instead of a direct measurement of the reaction rate, an indirect procedure was performed by comparing the effect of these amino acids with the reaction rate of dansylglycine and Tau-NHBr. Dansylglycine is a fluorescent probe used for attribution of binding sites in albumin [39]. This probe was selected for several reasons, including our previous determination of its bimolecular rate constant with HOBr and HOCl [15], its fluorescence properties (λ ex 360 nm, λ em 550 nm), which made the spectral interference of the studied compounds minimal, and its application as a probe for tryptophan residues in albumin, as will be demonstrated below.

Relative Reactivity with Tryptophan, Tyrosine, Cysteine and Methionine
Following the analytical protocol established above, the selectivity of Tau-NHBr with the oxidizable amino acids was evaluated by measuring and comparing tryptophan, tyrosine, cysteine and methionine in regard to their efficacy as inhibitors of the oxidation of dansylglycine. The results displayed in Figure 4 show the effect of tryptophan and tyrosine in the fluorescence bleaching of dansylglycine. It can be observed that, whereas tryptophan was an effective competitor and significantly inhibited the depletion of dansylglycine, tyrosine was much less effective. From these experiments, the relative reactivity of the amino acids was calculated as the slope of the curve (k obs versus amino acid concentration). The results were tryptophan 2.4ˆ10´4 ∆k obs /mM ( Figure 4b) and tyrosine 1.3ˆ10´5 ∆k obs /mM (Figure 4d), showing that tryptophan was about an 18-fold better inhibitor, or, in other words, 18-fold more reactive with Tau-NHBr compared with tyrosine.
Following the same experimental concept, the effects of cysteine and methionine on dansylglycine bleaching were also measured. The slope of the curve of the pseudo first-order rate constant versus amino acid concentration was cysteine 8.7ˆ10´5 ∆k obs /mM and methionine 6.5ˆ10´5 ∆k obs /mM ( Figure 5). Hence, the following sequence of relative reactivity of Tau-NHBr was established: tryptophan > cysteine~methionine > tyrosine. The results displayed in Figure 4 show the effect of tryptophan and tyrosine in the fluorescence bleaching of dansylglycine. It can be observed that, whereas tryptophan was an effective competitor and significantly inhibited the depletion of dansylglycine, tyrosine was much less effective. From these experiments, the relative reactivity of the amino acids was calculated as the slope of the curve (kobs versus amino acid concentration). The results were tryptophan 2.4 × 10 −4 ∆kobs/mM ( Figure 4b) and tyrosine 1.3 × 10 −5 ∆kobs/mM (Figure 4d), showing that tryptophan was about an 18-fold better inhibitor, or, in other words, 18-fold more reactive with Tau-NHBr compared with tyrosine.
Following the same experimental concept, the effects of cysteine and methionine on dansylglycine bleaching were also measured. The slope of the curve of the pseudo first-order rate constant versus amino acid concentration was cysteine 8.7 × 10 −5 ∆kobs/mM and methionine 6.5 × 10 −5 ∆kobs/mM ( Figure 5). Hence, the following sequence of relative reactivity of Tau-NHBr was established: tryptophan > cysteine ~ methionine > tyrosine.

Selectivity upon Tryptophan Residues in Proteins
As we have demonstrated above, Tau-NHBr has lower oxidant capacity compared to its precursor HOBr and HOCl. In this context, it is worth noting a principle of chemistry: lower reactivity usually implies greater selectivity. This principle seems to be applicable in our studies, because, in contrast to HOBr and HOCl, Tau-NHBr was able to oxidize tryptophan but not tyrosine. Following this idea, we suspect that our recent proposal for the selectivity of Tau-NHBr [40] and Tau-NBr2 [41] for tryptophan residues in albumin and lysozyme could be reinforced by these new findings. Thus, aiming to advance this proposal, dansylglycine was used to probe the depletion of tryptophan in human serum albumin (HSA).
Dansylglycine is a ligand of albumin. Its complexation can be monitored by an increased fluorescence quantum yield and a blue shift in the emission band, and, more importantly for our purpose, dansylated amino acids can be excited by fluorescence resonance energy transfer from tryptophan in HSA [42]. To make sure that this property is also applicable to dansylglycine, we added increasing concentrations of this compound to a fixed concentration of HSA. Our expectation was confirmed because dansylglycine was not fluorescent when excited at 295 nm; however, in the presence of HSA, the addition of increasing amounts of dansylglycine provoked a gradual quenching of the intrinsic fluorescence of the protein and a concomitant increase in the fluorescence of dansylglycine.
Considering these findings, dansylglycine was used as a probe for evaluation of the consumption of tryptophan in HSA provoked oxidation. In these experiments, the oxidation was provoked by adding a 20-fold excess of the oxidants and, after five minutes, methionine was added to deplete the excess of oxidants. The results depicted in Figure 6a confirmed our expectation of selectivity because, though much less reactive with free tryptophan, Tau-NHBr was more effective

Selectivity upon Tryptophan Residues in Proteins
As we have demonstrated above, Tau-NHBr has lower oxidant capacity compared to its precursor HOBr and HOCl. In this context, it is worth noting a principle of chemistry: lower reactivity usually implies greater selectivity. This principle seems to be applicable in our studies, because, in contrast to HOBr and HOCl, Tau-NHBr was able to oxidize tryptophan but not tyrosine. Following this idea, we suspect that our recent proposal for the selectivity of Tau-NHBr [40] and Tau-NBr 2 [41] for tryptophan residues in albumin and lysozyme could be reinforced by these new findings. Thus, aiming to advance this proposal, dansylglycine was used to probe the depletion of tryptophan in human serum albumin (HSA).
Dansylglycine is a ligand of albumin. Its complexation can be monitored by an increased fluorescence quantum yield and a blue shift in the emission band, and, more importantly for our purpose, dansylated amino acids can be excited by fluorescence resonance energy transfer from tryptophan in HSA [42]. To make sure that this property is also applicable to dansylglycine, we added increasing concentrations of this compound to a fixed concentration of HSA. Our expectation was confirmed because dansylglycine was not fluorescent when excited at 295 nm; however, in the presence of HSA, the addition of increasing amounts of dansylglycine provoked a gradual quenching of the intrinsic fluorescence of the protein and a concomitant increase in the fluorescence of dansylglycine.
Considering these findings, dansylglycine was used as a probe for evaluation of the consumption of tryptophan in HSA provoked oxidation. In these experiments, the oxidation was provoked by adding a 20-fold excess of the oxidants and, after five minutes, methionine was added to deplete the excess of oxidants. The results depicted in Figure 6a confirmed our expectation of selectivity because, though much less reactive with free tryptophan, Tau-NHBr was more effective than HOCl or HOBr in depleting the intrinsic fluorescence of HSA, which is mainly due to tryptophan residues when excited at 295 nm [43]. In others words, Tau-NHBr seems to act mainly on tryptophan residues of the protein. Figure 6b shows the effect of the addition of dansylglycine after oxidation and depletion of the oxidant excess. The band at 485 nm was lower in the sample oxidized with Tau-NHBr, which is an additional confirmation that tryptophan residues were more efficiently oxidized using this oxidant, since the measured fluorescence was due to energy transfer from the tryptophan residues in HSA.
Biomolecules 2016, 6, 23 7 of 17 than HOCl or HOBr in depleting the intrinsic fluorescence of HSA, which is mainly due to tryptophan residues when excited at 295 nm [43]. In others words, Tau-NHBr seems to act mainly on tryptophan residues of the protein. Figure 6b shows the effect of the addition of dansylglycine after oxidation and depletion of the oxidant excess. The band at 485 nm was lower in the sample oxidized with Tau-NHBr, which is an additional confirmation that tryptophan residues were more efficiently oxidized using this oxidant, since the measured fluorescence was due to energy transfer from the tryptophan residues in HSA.

Comparison between Tau-NHCl and Tau-NHBr
As we have demonstrated above, Tau-NHCl was unreactive with all studied compounds in this work; which is, indeed, in agreement with its well-established poor oxidant capacity [32]. Obviously, it does not mean that Tau-NHCl is completely devoid of oxidant capacity, as is confirmed, for instance, by its capacity to oxidize sulfhydryl residues in proteins [27]. Tau-NHCl is also able to oxidize 3,3',5,5'-tetramethylbenzidine (TMB) in acid medium, the chromogenic substrate used for determination of the chlorination activity of MPO [44]. Thus, aiming to quantify the difference in reactivity between Tau-NHBr and Tau-NHCl, we used TMB as the target. It must also be noted that instead of an acidic pH, the reaction was conducted at pH 7.0, as was used for the other compound studies in this work. It is important to emphasize this point, because its reactivity is significantly lower at neutral pH, which is, indeed, the reason for the application of acidic medium for determination of Tau-NHCl activity using TMB [44]. The results depicted in Figure 7 confirmed our expectation, because, at pH 7.0, Tau-NHCl was able to oxidize TMB. Its oxidant efficacy was measured (k2 = 3.1 M −1 s −1 ) and, corroborant with the previous results, was about 100-fold lower than Tau-NHBr (k2 = 6.4 × 10 2 M −1 s −1 ). The comparison was also made using the antioxidant curcumin, an

Comparison between Tau-NHCl and Tau-NHBr
As we have demonstrated above, Tau-NHCl was unreactive with all studied compounds in this work; which is, indeed, in agreement with its well-established poor oxidant capacity [32]. Obviously, it does not mean that Tau-NHCl is completely devoid of oxidant capacity, as is confirmed, for instance, by its capacity to oxidize sulfhydryl residues in proteins [27]. Tau-NHCl is also able to oxidize 3,3',5,5'-tetramethylbenzidine (TMB) in acid medium, the chromogenic substrate used for determination of the chlorination activity of MPO [44]. Thus, aiming to quantify the difference in reactivity between Tau-NHBr and Tau-NHCl, we used TMB as the target. It must also be noted that instead of an acidic pH, the reaction was conducted at pH 7.0, as was used for the other compound studies in this work. It is important to emphasize this point, because its reactivity is significantly lower at neutral pH, which is, indeed, the reason for the application of acidic medium for determination of Tau-NHCl activity using TMB [44]. The results depicted in Figure 7 confirmed our expectation, because, at pH 7.0, Tau-NHCl was able to oxidize TMB. Its oxidant efficacy was measured (k 2 = 3.1 M´1s´1) and, corroborant with the previous results, was about 100-fold lower than Tau-NHBr (k 2 = 6.4ˆ10 2 M´1s´1). The comparison was also made using the antioxidant curcumin, an oxidizable

pH Effect on Tau-NHBr Reactivity
In aqueous solutions, chloramine and bromamine are in equilibrium with their dichloramine and dibromamine forms [32,33]. These dihalogenated structures are the results of the disproportionation reaction, by which two molecules of Tau-NHX are converted to Tau-NX 2 and taurine, respectively. The conversion of Tau-NHCl to Tau-NHCl 2 is pH dependent, being favored at acidic pH [32]. Here, we found the same tendency for Tau-NHBr, which was progressively, converted to Tau-NBr 2 as the pH decreased. The results depicted in Figure 8a show the absorbance increase at 241 and 346 nm and decrease at 288 nm, which provides evidence for the formation of Tau-NBr 2 [33]. The chemical equation below shows the equilibrium between the mono-and dihalogenated forms as well as the pH dependence (Equation 2).

pH Effect on Tau-NHBr Reactivity
In aqueous solutions, chloramine and bromamine are in equilibrium with their dichloramine and dibromamine forms [32,33]. These dihalogenated structures are the results of the disproportionation reaction, by which two molecules of Tau-NHX are converted to Tau-NX2 and taurine, respectively. The conversion of Tau-NHCl to Tau-NHCl2 is pH dependent, being favored at acidic pH [32]. Here, we found the same tendency for Tau-NHBr, which was progressively, converted to Tau-NBr2 as the pH decreased. The results depicted in Figure 8a show the absorbance increase at 241 and 346 nm and decrease at 288 nm, which provides evidence for the formation of Tau-NBr2 [33]. The chemical equation below shows the equilibrium between the mono-and dihalogenated forms as well as the pH dependence (Equation 2).
2 HO 3 SCH 2 CH 2 NHBr + H + ↔ HO 3 SCH 2 CH 2 NBr 2 + HO 3 SCH 2 CH 2 NH 3 Next, we studied the effect of pH on the reactivity of Tau-NHBr. Figure 8 shows the k2 obtained for the reactions between dansylglycine and Tau   Next, we studied the effect of pH on the reactivity of Tau-NHBr. Figure 8 shows the k 2 obtained for the reactions between dansylglycine and Tau-NHBr at pH 5.0 and 9.0. The rate constants were (pH 5.0) 1.1ˆ10 4 M´1s´1, (pH 7.0) 9.5ˆ10 1 M´1s´1 and (pH 9.0) 1.7ˆ10 1 M´1s´1. The pH dependence can be explained by taking into account the higher reactivity of the dihalogenated forms, as has been demonstrated for Tau-NCl 2 [45].

Reactivity of Tau-NHBr with Hydrogen Peroxide and Formation of Singlet Oxygen
Another property that distinguishes Tau-NHCl from Tau-NHBr is the capacity of the latter to react with H 2 O 2 [33]. Here, this chemical property was confirmed and the k 2 (3.9 M´1s´1) obtained by measuring the decay of Tau-NHBr as a function of increasing concentrations of H 2 O 2 (Figure 9). Activated eosinophils are an important in vivo source of singlet oxygen. The formation of this electronically excited form of oxygen is due to the reaction between HOBr and H2O2 [46]. We found that Tau-NHBr retains this capacity, as can be seen by the ultra-weak light emission produced during the reaction course originating from the decay of singlet oxygen (Figure 10a). It is important to emphasize that the extremely low light emission is due to the inadequacy of a conventional luminometer to measure the emission of singlet oxygen, which emits in the infrared region (1200 nm). Hence, to gain additional evidence of its production, we added melatonin to the reaction system. The results depicted in Figure 10b show that the light emission increased almost two orders of magnitude compared to the reaction without melatonin. The light emission is dependent on both H2O2 and melatonin, which excluded the possibility of direct interaction between melatonin and Tau-NHBr or melatonin and H2O2 as a source of the light emission. Thus, the increase in emission is consistent with the cleavage of the pyrrole ring of melatonin and formation of N-acetyl-N-formyl-5methoxykynuramine (AFMK), a chemiluminescent reaction [47] that has been demonstrated between melatonin and singlet oxygen [48]. In accordance with this proposal, the formation of AFMK was also obtained here (Figure 10c).

Reactivity of Tau-NHBr with Linoleic Acid
Among the biomolecules susceptible to the deleterious effects provoked by reactive oxygen species (ROS), unsaturated fatty acids occupy an important position. As electrophilic reagents, HOCl and HOBr are well established as inducers of the formation of halohydrin by reacting with the double bonds in mono-and polyunsaturated fatty acids [49,50]. For this reason, we also investigated the reactivity of Tau Activated eosinophils are an important in vivo source of singlet oxygen. The formation of this electronically excited form of oxygen is due to the reaction between HOBr and H 2 O 2 [46]. We found that Tau-NHBr retains this capacity, as can be seen by the ultra-weak light emission produced during the reaction course originating from the decay of singlet oxygen (Figure 10a). It is important to emphasize that the extremely low light emission is due to the inadequacy of a conventional luminometer to measure the emission of singlet oxygen, which emits in the infrared region (1200 nm). Hence, to gain additional evidence of its production, we added melatonin to the reaction system. The results depicted in Figure 10b show that the light emission increased almost two orders of magnitude compared to the reaction without melatonin. The light emission is dependent on both H 2 O 2 and melatonin, which excluded the possibility of direct interaction between melatonin and Tau-NHBr or melatonin and H 2 O 2 as a source of the light emission. Thus, the increase in emission is consistent with the cleavage of the pyrrole ring of melatonin and formation of N-acetyl-N-formyl-5-methoxykynuramine (AFMK), a chemiluminescent reaction [47] that has been demonstrated between melatonin and singlet oxygen [48]. In accordance with this proposal, the formation of AFMK was also obtained here (Figure 10c). experiments, linoleic acid was incubated with the oxidants and their remaining concentration was measured using the sulfhydryl reagent 5'-thio-2-nitrobenzoic acid (TNB). The results depicted in Figure 11 show that, while HOCl and HOBr reacted promptly with linoleic acid and were totally consumed just after one minute of incubation, Tau-NHBr reacted slowly, with only 50% consumed in 60 min of incubation. As could be expected from the previous results, Tau-NHCl was still less reactive.

Reactivity of Tau-NHBr with Linoleic Acid
Among the biomolecules susceptible to the deleterious effects provoked by reactive oxygen species (ROS), unsaturated fatty acids occupy an important position. As electrophilic reagents, HOCl and HOBr are well established as inducers of the formation of halohydrin by reacting with the double bonds in mono-and polyunsaturated fatty acids [49,50]. For this reason, we also investigated the reactivity of Tau-NHBr with linoleic acid as a model of polyunsaturated fatty acids. In these experiments, linoleic acid was incubated with the oxidants and their remaining concentration was measured using the sulfhydryl reagent 5'-thio-2-nitrobenzoic acid (TNB). The results depicted in Figure 11 show that, while HOCl and HOBr reacted promptly with linoleic acid and were totally consumed just after one minute of incubation, Tau-NHBr reacted slowly, with only 50% consumed in 60 min of incubation. As could be expected from the previous results, Tau-NHCl was still less reactive.

Reactivity of Tau-NHBr with Nucleosides
Other well established endogenous targets for hypohalous acids are nucleosides, nucleotides, DNA and RNA. For instance, 5-chlorouracil and 5-bromouracil are formed from the MPO catalyzed oxidation of uracil, and 5-chloro-2'-deoxycytidine, 8-chloro-2'-deoxyadenosine and 8-chloro-2'deoxyguanosine result from the treatment of DNA with HOCl [51]. Recently, the miscoding properties of 8-chloro-2'-deoxyguanosine have been demonstrated, which highlights the potential importance of HOCl-mediated reactions in the pathogenesis of inflammation-driven carcinogenesis [52]. Similarly, the presence of 8-bromo-2'-deoxyguanosine has been proposed as a substance that may increase mutagenic potential at the site of inflammation [53]. For this reason, we also evaluated the reactivity of Tau-NHBr with nucleosides. However, the superposition of absorption spectra between Tau-NHBr, and the nucleosides impeded the direct measurement of their consumption. Hence, we again employed an indirect experimental approach by incubating the nucleosides with the oxidant and then measuring the remaining oxidant with the TNB reagent. The results depicted in Figure 12 show that, in contrast to HOBr, Tau-NHBr was significantly less reactive with nucleosides.

Reactivity of Tau-NHBr with Nucleosides
Other well established endogenous targets for hypohalous acids are nucleosides, nucleotides, DNA and RNA. For instance, 5-chlorouracil and 5-bromouracil are formed from the MPO catalyzed oxidation of uracil, and 5-chloro-2'-deoxycytidine, 8-chloro-2'-deoxyadenosine and 8-chloro-2'-deoxyguanosine result from the treatment of DNA with HOCl [51]. Recently, the miscoding properties of 8-chloro-2'-deoxyguanosine have been demonstrated, which highlights the potential importance of HOCl-mediated reactions in the pathogenesis of inflammation-driven carcinogenesis [52]. Similarly, the presence of 8-bromo-2'-deoxyguanosine has been proposed as a substance that may increase mutagenic potential at the site of inflammation [53]. For this reason, we also evaluated the reactivity of Tau-NHBr with nucleosides. However, the superposition of absorption spectra between Tau-NHBr, and the nucleosides impeded the direct measurement of their consumption. Hence, we again employed an indirect experimental approach by incubating the nucleosides with the oxidant and then measuring the remaining oxidant with the TNB reagent. The results depicted in Figure 12 show that, in contrast to HOBr, Tau-NHBr was significantly less reactive with nucleosides.

Reactivity of Tau-NHBr with Nucleosides
Other well established endogenous targets for hypohalous acids are nucleosides, nucleotides, DNA and RNA. For instance, 5-chlorouracil and 5-bromouracil are formed from the MPO catalyzed oxidation of uracil, and 5-chloro-2'-deoxycytidine, 8-chloro-2'-deoxyadenosine and 8-chloro-2'deoxyguanosine result from the treatment of DNA with HOCl [51]. Recently, the miscoding properties of 8-chloro-2'-deoxyguanosine have been demonstrated, which highlights the potential importance of HOCl-mediated reactions in the pathogenesis of inflammation-driven carcinogenesis [52]. Similarly, the presence of 8-bromo-2'-deoxyguanosine has been proposed as a substance that may increase mutagenic potential at the site of inflammation [53]. For this reason, we also evaluated the reactivity of Tau-NHBr with nucleosides. However, the superposition of absorption spectra between Tau-NHBr, and the nucleosides impeded the direct measurement of their consumption. Hence, we again employed an indirect experimental approach by incubating the nucleosides with the oxidant and then measuring the remaining oxidant with the TNB reagent. The results depicted in Figure 12 show that, in contrast to HOBr, Tau-NHBr was significantly less reactive with nucleosides.

Determination of Rate Constants
The reactivity of Tau-NHBr, HOBr and HOCl with the target molecules was obtained by comparing their bimolecular rate constants, which were obtained using pseudo-first-order experimental conditions. The fast-kinetic experiments were performed using a single-mixing stopped-flow system equipped with a high intensity LED source and cut-off filters (SX20/LED Stopped-Flow System, Applied Photophysics, City, UK). The observed pseudo-first-order rate constant (k obs ) was obtained by fitting the fluorescence or absorbance decay of the studied compound to a single exponential decay equation, as follows (Equation 3): where: S is the fluorescence or absorbance as a function of time S 0 is the initial fluorescence or absorbance From the k obs values obtained at various concentrations, the bimolecular rate constants (k 2 ) were calculated from the slope of the linear regression as follows (Equation 4): where [A] is the concentration of the haloamines or hypohalous acids and [B] is the concentration of the studied compounds. From the pseudo-first-order experimental condition, the apparent second-order rate constant (k 2 ) was calculated (Equation 5).
If rAs " rBs, then, reaction rate " k obsˆr Bs Then, k obs " k 2ˆr As, The k 2 is the slope of the linear fit of k obs versus [A] The photophysical properties of the studied compounds and their intrinsic reactivity determined the experimental conditions used to monitor each reaction. The specific absorbance or excitation and emission wavelengths used for monitoring each reaction are shown in the figure legends. Unless otherwise stated, the concentrations of the target compounds were fixed at 25 µM, and the concentrations of the oxidants were in the range of 100 to 500 µM. The reactions were performed in 50 mM phosphate buffer, pH 7.0 at 25˝C.

Oxidation of Human Albumin and the Use of Dansylglycine as a Fluorescent Probe for Tryptophan Residues
The reaction mixtures were composed of 10 µM HSA and 200 µM oxidants in 50 mM phosphate buffer, pH 7.0. After five minutes, 250 µM methionine was added and the fluorescence was measured at an excitation wavelength of 295 nm and emission in the range of 310-450 nm was recorded. When present, dansylglycine was added at 5 µM and emission was measured in the range of 450-600 nm. The experiments were performed using a LS55 spectrofluorometer (Perkin-Elmer, Waltham, MA, USA). For the experiments where HSA was oxidized in the presence of dansylglycine, the stopped-flow system was set as follows: excitation, 280 nm LED, and emission, 455 nm cut-off filter.
Curcumin: The reaction medium was composed of 10 µM curcumin and increasing concentrations of haloamines in 50 mM phosphate buffer, pH 7.0. The reaction was monitored at 425 nm using conventional spectrophotometry.
Hydrogen peroxide: The reaction medium was composed of 250 µM Tau-NHBr and increasing concentrations of H 2 O 2 in 50 mM phosphate buffer, pH 7.0. The reaction was monitored at 288 nm using conventional spectrophotometry. The reaction also was studied by chemiluminescence. In this case, the measurement of light emission was performed using a Centro LB 960 microplate luminometer (Berthold Technologies, Oak Ridge, TN, USA). The isolation and identification of oxidation products of melatonin was performed by high performance liquid chromatography in line with a fluorescence detector set at 340/460 nm (Jasco, Easton, MD, USA). The analyses were carried out isocratically on a Luna C18 reverse-phase column (250ˆ4.6 mm, 5 µm) using 70:30 aqueous formic acid 0.1%/acetonitrile (flow rate 1.0 mL/min) as the mobile phase. The identification of AFMK as a product of the reaction was performed by comparison with a pure standard of this compound.
Linoleic acid: The oxidants (50 µM) were incubated with 100 µM linoleic acid in 50 mM phosphate buffer, pH 7.0 for increasing time intervals. Then, the remaining concentration of oxidants was measured by the addition of 50 µM TNB, and the absorbance was measured at 412 nm (ε 412nm = 14,100 M´1cm´1).

Conclusions
Tau-NHBr, an endogenous oxidant, has also been used as an anti-inflammatory compound and topical antimicrobial agent. Here, we have identified several chemical properties of this promising drug. The most important was its selectivity regarding tryptophan residues in proteins. By measuring the bimolecular rate constant with several biomolecules, the following reactivity sequence was established: HOBr > HOCl > Tau-NHBr > Tau-NHCl. The reactivity of Tau-NHBr was strongly affected by the pH of the medium, a property related to the formation of the dibromamine form at acidic pH (Tau-NBr2). The formation of singlet oxygen was observed in the reaction between Tau-NHBr and H2O2. In conclusion, these findings could be very helpful for researchers interested in biological applications of this taurine haloamine.