biomolecules Article

Taurine Bromamine: Reactivity of an Endogenous and Exogenous Anti-Inflammatory and Antimicrobial Amino Acid Derivative Luiza De Carvalho Bertozo 1 , Nelson Henrique Morgon 2 , Aguinaldo Robinson De Souza 1 and Valdecir Farias Ximenes 1, * 1 2

*

Department of Chemistry, Faculty of Sciences, São Paulo State University (UNESP), Bauru 17033-360, Brazil; [email protected] (L.D.C.B.); [email protected] (A.R.D.S.) Department of Chemistry, Institute of Chemistry, Campinas State University (UNICAMP), Campinas 13083-861, Brazil; [email protected] Correspondence: [email protected]; Tel.: +55-014-3103-6088

Academic Editor: Jürg Bähler Received: 27 March 2016; Accepted: 13 April 2016; Published: 21 April 2016

Abstract: 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 ˆ 102 M´1 s´1 ), melatonin (7.3 ˆ 103 M´1 s´1 ), serotonin (2.9 ˆ 103 M´1 s´1 ), dansylglycine (9.5 ˆ 101 M´1 s´1 ), tetramethylbenzidine (6.4 ˆ 102 M´1 s´1 ) and H2 O2 (3.9 ˆ M´1 s´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 ˆ 104 M´1 s´1 , pH 7.0, 9.5 ˆ 10 M´1 s´1 and pH 9.0, 1.7 ˆ 10 M´1 s´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 H2 O2 . 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. Keywords: taurine bromamine; tryptophan; melatonin; serotonin; singlet oxygen; nucleosides

1. 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 Biomolecules 2016, 6, 23; doi:10.3390/biom6020023

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transient production of the redox active compounds, named compound I (MPO-I and EPO-I), which are two electron oxidized compared with the native enzyme. An important difference between MPO-I and EPO-I is their standard reduction potential [10]. Thus, while MPO-I (1.16 V) is able to oxidize efficiently both Cl´ and Br´ [11]; EPO-I (1.09 V) shows a large preference for Br´ [12]. This chemical feature, the higher plasma level of Cl´ (~100 mM) compared with Br´ (~100 µM), and the higher oxidant capacity of HOCl (1.28 V) compared with HOBr (1.13 V) [13], could suggest that the biological effect of HOBr would be irrelevant compared with HOCl. However, this does not seem to be the case, since there is significant evidence that HOBr is more reactive with several biomolecules compared to HOCl [14,15]. This phenomenon is the consequence of the higher electrophilicity of HOBr compared to HOCl [16]. Taurine bromamine (Tau-NHBr) is a chemical produced by the reaction between HOBr and the non-essential amino acid taurine. Yazdanbakhsh et al. provided the first application of Tau-NHBr and proposed its endogenous formation [17]. These authors found that stimulated eosinophils could be a source of Tau-NHBr, since these cells may produce HOBr, and taurine is abundantly present in leukocytes. It is interesting to note that this finding took place just one year after the discovery that eosinophils could produce and release HOBr [18]. Currently, Tau-NHBr is used as an anti-inflammatory and a topical antimicrobial drug. Some examples of its applications are its use as a therapeutic agent for the treatment of acne vulgares [19,20]; treatment of biofilm-associated infections on dental surfaces caused by Pseudomonas aeruginosa [21,22]; microbicidal activity against Escherichia coli and Staphylococcus aureus at insensitive body regions with low organic matter [23]; inhibition of the production of inflammatory mediators, such as prostaglandin E2 (PGE2), nitric oxide (NO) and pro-inflammatory cytokines [24]; and inhibition of degradation of TNF-α-induced Nuclear Factor-kappaB activation in Jurkat cells and myeloid-committed eosinophils [25]. The equivalent chlorinated haloamine is taurine chloramine (Tau-NHCl), which is the reaction product of the interaction between taurine and HOCl. Tau-NHCl is produced by activated neutrophils and released at inflammatory sites, inhibiting the production of inflammatory mediators [26]. An important chemical feature of Tau-NHCl, which makes this compound so interesting and extensively studied, is its mild oxidant capacity compared to its precursor HOCl. Thus, Tau-NHCl is able to oxidize selectively sulfhydryl residues in proteins [27], and to act as an endogenous antioxidant [28]. These aspects might be involved in the signaling pathways susceptible to Tau-NHCl [29,30]. There are also a large number of applications for Tau-NHCl as a topical anti-inflammatory and anti-infective drug [31]. In contrast to Tau-NHCl, for which the chemical properties have been intensively studied [32], much less is known about Tau-NHBr. This was our motivation for undertaking this study. Here, the reactivity of Tau-NHBr was evaluated and compared with HOBr, HOCl and Tau-NHCl, using several endogenous and non-endogenous chemicals. Using fast kinetic techniques, it was possible to measure the bimolecular rate constants of these reactions. We believe that this chemical data will be helpful for those interested in the application of this interesting compound. 2. Results and Discussion 2.1. 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-NBr2 ) 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]. HO3 SCH2 CH2 NH2 pTaurineq

` HOBr Ñ

HO3 SCH2 CH2 NHBr pTau ´ NHBrq,

(1)

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2.2. 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 (k2 ) was calculated (7.7 ˆ 102 M´1 s´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 k2 ; 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 (k2 = 8.1ˆ104 M´1 s´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 k2 values obtained for melatonin (7.3 ˆ 103 M´1 s´1 ) and serotonin (2.9 ˆ 103 M´1 s´1 ) were about 10-fold higher compared to tryptophan (Figure 2). 2.3. 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 k2 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 (λex 360 nm, λem 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. Before the use of dansylglycine for the comparison of the reactivity of these amino acids, the measurement of its apparent second-order rate constant with Tau-NHBr was performed (Figure 3). Corroborant with the tryptophan results, the obtained k2 (9.5 ˆ 101 M´1 s´1 ) was significantly lower than for HOBr (7.3 ˆ 106 M´1 s´1 ) and HOCl (5.2 ˆ 102 M´1 s´1 ) [15]. Tau-NHCl was totally unreactive with dansylglycine. The following reactivity sequence again was established: HOBr > HOCl > Tau-NHBr > Tau-NHCl. 2.4. 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.

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4 of 17 The results displayed in Figure 4 show the effect of tryptophan and tyrosine in the fluorescence bleachingThe of dansylglycine. be observed tryptophan was an competitor results displayedItincan Figure 4 show thethat, effectwhereas of tryptophan and tyrosine in effective the fluorescence and significantly inhibited theIt depletion of dansylglycine, tyrosine was less effective. bleaching of dansylglycine. can be observed that, whereas tryptophan was much an effective competitorFrom theseand experiments, relativethe reactivity amino acidstyrosine was calculated theeffective. slope ofFrom the curve significantlythe inhibited depletionofofthe dansylglycine, was muchasless ´ 4 ∆k /mM (Figure 4b) (kobs versus amino acid concentration). The results were tryptophan 2.4 ˆ 10 these experiments, the relative reactivity of the amino acids was calculated as the slope obs of the curve ´5 ∆k (kobs versus1.3 amino concentration). The results were tryptophan 2.4 × 10 −4 ∆k obs/mM (Figure 4b) andbetter and tyrosine ˆ 10acid 4d), showing that tryptophan was about an 18-fold obs /mM (Figure tyrosine 1.3other × 10−5words, ∆kobs/mM (Figure 4d), showing thatTau-NHBr tryptophan was about an tyrosine. 18-fold better inhibitor, or, in 18-fold more reactive with compared with inhibitor, in other 18-fold more reactive with Tau-NHBr compared with tyrosine. Followingor,the same words, experimental concept, the effects of cysteine and methionine on dansylglycine Following the same experimental concept, the effects of cysteine and methionine on bleaching were also measured. The slope of the curve of the pseudo first-order rate constant versus dansylglycine bleaching were also measured. The slope of the curve of the pseudo first-order rate amino acid concentration was cysteine 8.7 ˆ 10´5 ∆kobs /mM and methionine 6.5 ˆ 10´5 ∆k −5/mM constant versus amino acid concentration was cysteine 8.7 × 10 −5 ∆kobs/mM and methionine 6.5 × 10obs (Figure relativeofreactivity of Tau-NHBr was established: ∆kobs5). /mMHence, (Figure the 5). following Hence, the sequence following of sequence relative reactivity of Tau-NHBr was tryptophan > cysteine ~ methionine > tyrosine. established: tryptophan > cysteine ~ methionine > tyrosine.

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Figure 1. Reactivity of Tau-NHBr and HOBr with tryptophan. (a) Kinetic profile of tryptophan

Figure 1. Reactivity of Tau-NHBr and HOBr with tryptophan. (a) Kinetic profile of tryptophan consumption for determination of kobs under pseudo-first-order experimental conditions. The reaction consumption for determination of kobs under pseudo-first-order experimental conditions. The reaction mixture was composed of 25 µ M tryptophan and increasing concentrations of Tau-NHBr in 50 mM mixture was composed of 25 µM tryptophan and increasing concentrations of Tau-NHBr in 50 mM phosphate buffer, pH 7.0 at 25 °C. The stopped-flow system was set as follows: excitation, 280 nm ˝ C. The stopped-flow system was set as follows: excitation, 280 nm phosphate buffer, pH 7.0 25cut-off LED; and emission, 325at nm filter; (b) Determination of the bimolecular rate constant (k2) for LED;the andreaction emission, 325 nmtryptophan cut-off filter; (b)Tau-NHBr; Determination of the bimolecular rate constant ) for the between and (c) Kinetic profile of tryptophan (25 (kµ2M) reaction between by tryptophan (c) the Kinetic ofexperiments. tryptophan (25 µM) consumption by consumption HOBr (50 and µ M).Tau-NHBr; The results are meanprofile of three HOBr (50 µM). The results are the mean of three experiments.

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Biomolecules 2016, a) 6, 23 Tau-NHBr 194 M

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200 300 400 500 2 3 4 5 0.4 Tau-NHBr (M) Time (s) 0 200 300 400 500 0 1 2 3 4 5 Figure 2. Reactivity of Tau-NHBr with melatonin and serotonin. Kinetic profile of (a) Melatonin and Tau-NHBr (M) Time (s) Figure 2. Reactivity of Tau-NHBr with melatonin and serotonin. Kinetic profile of (a) Melatonin and

1

(c) Serotonin consumption for determination of kobs under pseudo-first-order experimental conditions. (c) Figure Serotonin consumption for determination of kobsand under pseudo-first-order experimental conditions. 2. Reactivity Tau-NHBr with melatonin serotonin. Kinetic profile of (a)ofMelatonin The reaction mixtureofwas composed of 25 µ M indoles and increasing concentrations Tau-NHBrand in The(c)reaction mixture was composed of 25 µM indoles and increasing concentrations of Tau-NHBr in Serotonin consumption for determination of k obs under pseudo-first-order experimental conditions. 50 mM phosphate buffer, pH 7.0 at 25 °C. The stopped-flow system was set as follows: excitation, 280 ˝ C. The stopped-flow system was set as follows: excitation, 50 The mMreaction phosphate buffer, pH 7.0 at 25 was composed of 25 µ MDetermination indoles and increasing concentrations of Tau-NHBr in nm LED; andmixture emission, 325 nm cut-off filter. of the bimolecular rate constant (k2) for 28050nm LED; and emission, 3257.0 nm cut-off filter. Determination of was the bimolecular rate constant (k2 ) mM phosphate buffer, pH at 25 °C. The stopped-flow system set as follows: excitation, 280 the reaction between (b) Melatonin and (d) Serotonin with Tau-NHBr. The results are the mean of fornm theLED; reaction between (b) Melatonin and (d)Determination Serotonin withofTau-NHBr. The results are the(kmean and emission, 325 nm cut-off filter. the bimolecular rate constant 2) for of three experiments. three theexperiments. reaction between (b) Melatonin and (d) Serotonin with Tau-NHBr. The results are the mean of three experiments.

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Figure 3. Reactivity of Tau-NHBr with dansylglycine. (a) Kinetic profile of dansylglycine Tau-NHBr (M) consumption for determination of kobs under pseudo-first-order experimental conditions. The reaction Figure was 3. Reactivity of Tau-NHBr with dansylglycine. (a) Kinetic profile ofof dansylglycine mixture composed of 25 µ Mwith dansylglycine (DG) (a) andKinetic increasing concentrations Tau-NHBr in Figure 3. Reactivity of Tau-NHBr dansylglycine. profile of dansylglycine consumption consumption for determination of k obs under pseudo-first-order experimental conditions. The reaction mM phosphateofbuffer, pH 7.0pseudo-first-order at 25 °C. The stopped-flow system conditions. was set as follows: excitation, 360 for50determination kobs under experimental The reaction mixture mixture of475 25 µnm M dansylglycine (DG)Determination and increasingof concentrations of Tau-NHBr in nm LED;was andcomposed cut-off filter; k2 for of theTau-NHBr reaction between was composed ofemission, 25 µM dansylglycine (DG) and(b) increasing concentrations in 50 mM 50 mM phosphate buffer, pH 7.0 at 25 °C. The stopped-flow system was set as follows: excitation, 360 dansylglycine The results are the mean of three experiments. phosphate buffer,and pHTau-NHBr. 7.0 at 25 ˝ C. The stopped-flow system was set as follows: excitation, 360 nm LED; nm LED; and emission, 475 nm cut-off filter; (b) Determination of k2 for the reaction between and emission, 475 nm cut-off filter; (b) Determination of k2 for the reaction between dansylglycine and dansylglycine and Tau-NHBr. The results are the mean of three experiments. Tau-NHBr. The results are the mean of three experiments.

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Figure Tryptophanversus versus tyrosine forfor Tau-NHBr. (a) Reaction of dansylglycine with TauFigure 4. 4.Tryptophan tyrosineasastargets targets Tau-NHBr. (a) Reaction of dansylglycine with NHBr and the effect of tryptophan; (b) Effect of tryptophan on the pseudo-first-order reaction rate of Tau-NHBr and the effect of tryptophan; (b) Effect of tryptophan on the pseudo-first-order reaction dansylglycine with Tau-NHBr; (c) Reaction of dansylglycine with Tau-NHBr and the effect of rate of dansylglycine with Tau-NHBr; (c) Reaction of dansylglycine with Tau-NHBr and the effect of tyrosine; (d) Effect of tyrosine on the pseudo-first-order reaction rate of dansylglycine with Tautyrosine; (d) Effect of tyrosine on the pseudo-first-order reaction rate of dansylglycine with Tau-NHBr. NHBr. The reaction mixture was composed of 25 µ M dansylglycine, 500 µ M Tau-NHBr and The reaction mixture was composed of 25 µM dansylglycine, 500 µM Tau-NHBr and increasing increasing concentrations of amino acids in 50 mM phosphate buffer, pH 7.0. The stopped-flow concentrations of amino acids in 50 mM phosphate buffer, pH 7.0. The stopped-flow system was set as system was set as follows: excitation, 360 nm LED; and emission, 475 nm cut-off filter. follows: excitation, 360 nm LED; and emission, 475 nm cut-off filter.

2.5. Selectivity upon Tryptophan Residues in Proteins

2.5. Selectivity upon Tryptophan Residues in Proteins

As we have demonstrated above, Tau-NHBr has lower oxidant capacity compared to its

As we have demonstrated Tau-NHBr has lower capacity compared to its precursor precursor HOBr and HOCl. Inabove, this context, it is worth notingoxidant a principle of chemistry: lower reactivity HOBr and HOCl. In this context, it is worth noting a principle of chemistry: lower reactivity usually usually implies greater selectivity. This principle seems to be applicable in our studies, because, in implies greater selectivity. ThisTau-NHBr principle was seems toto beoxidize applicable in our studies, because,Following in contrast contrast to HOBr and HOCl, able tryptophan but not tyrosine. idea, weHOCl, suspectTau-NHBr that our recent for the selectivity of Tau-NHBr [40] and Tau-NBr 2 [41] to this HOBr and wasproposal able to oxidize tryptophan but not tyrosine. Following this for tryptophan residues in albumin and lysozyme could be reinforced by these new findings. Thus, idea, we suspect that our recent proposal for the selectivity of Tau-NHBr [40] and Tau-NBr2 [41] for aiming toresidues advanceinthis proposal, was to probe the depletion of tryptophan in tryptophan albumin and dansylglycine lysozyme could be used reinforced by these new findings. Thus, aiming human serum albumin (HSA). to advance this proposal, dansylglycine was used to probe the depletion of tryptophan in human Dansylglycine serum albumin (HSA).is a ligand of albumin. Its complexation can be monitored by an increased fluorescence quantum blue shift Its in the emission band, more importantly for our Dansylglycine is a yield ligandand of aalbumin. complexation can and, be monitored by an increased purpose, dansylated amino acids can be excited by fluorescence resonance energy transfer from fluorescence quantum yield and a blue shift in the emission band, and, more importantly for our tryptophan in HSA [42]. To make sure that this property is also applicable to dansylglycine, we added purpose, dansylated amino acids can be excited by fluorescence resonance energy transfer from increasing concentrations of this compound to a fixed concentration of HSA. Our expectation was tryptophan in HSA [42]. To make sure that this property is also applicable to dansylglycine, we added confirmed because dansylglycine was not fluorescent when excited at 295 nm; however, in the increasing concentrations of this compound to a fixed concentration of HSA. Our expectation was presence of HSA, the addition of increasing amounts of dansylglycine provoked a gradual quenching confirmed because dansylglycine was not fluorescent when excited at 295 nm; however, in the presence of the intrinsic fluorescence of the protein and a concomitant increase in the fluorescence of of HSA, the addition of increasing amounts of dansylglycine provoked a gradual quenching of the dansylglycine. intrinsicConsidering fluorescencethese of thefindings, protein and a concomitant in the fluorescence of dansylglycine. dansylglycine wasincrease used as a probe for evaluation of the Considering these findings, dansylglycine was used as a probe for evaluation of consumption consumption of tryptophan in HSA provoked oxidation. In these experiments, the the oxidation was of tryptophan HSA aprovoked oxidation. In theseand, experiments, the oxidation was provoked provoked byin adding 20-fold excess of the oxidants after five minutes, methionine was addedby adding a 20-fold oxidants after five minutes, methionine was added to depleteofthe to deplete the excess excess of of the oxidants. Theand, results depicted in Figure 6a confirmed our expectation excess of oxidants. The results depicted in Figure 6a confirmed our expectation of selectivity because, selectivity because, though much less reactive with free tryptophan, Tau-NHBr was more effective though much less reactive with free tryptophan, Tau-NHBr was more effective than HOCl or HOBr in

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than HOCl or HOBrfluorescence in depleting the fluorescence of HSA, which is mainlyresidues due to tryptophan depleting the intrinsic of intrinsic HSA, which is mainly due to tryptophan when excited residues when excited at 295 nm [43]. In others words, Tau-NHBr seems to act mainly on tryptophan at 295 nm [43]. In others words, Tau-NHBr seems to act mainly on tryptophan residues of the protein. residues of the protein. Figure 6b shows the effect of the addition of dansylglycine after oxidation Figure 6b shows the effect of the addition of dansylglycine after oxidation and depletion of the oxidant and depletion of the oxidant excess. The band at 485 nm was lower in the sample oxidized with Tauexcess. The band at 485 nm was lower in the sample oxidized with Tau-NHBr, which is an additional NHBr, which is an additional confirmation that tryptophan residues were more efficiently oxidized confirmation tryptophan residues were more efficiently usingfrom thisthe oxidant, since the using this that oxidant, since the measured fluorescence was due to oxidized energy transfer tryptophan measured fluorescence was due to energy transfer from the tryptophan residues in HSA. residues in HSA. without cysteine + cysteine 125 M // 250 // 315 // 375

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0

Figure 5. Cysteine versusmethionine methionine asastargets for for Tau-NHBr. (a) Reaction of dansylglycine with Tau- with Figure 5. Cysteine versus targets Tau-NHBr. (a) Reaction of dansylglycine NHBr and the effect of cysteine; (b) Effect of cysteine on the pseudo-first-order reaction rate of Tau-NHBr and the effect of cysteine; (b) Effect of cysteine on the pseudo-first-order reaction rate dansylglycine with Tau-NHBr; (c) Reaction of dansylglycine with Tau-NHBr and the effect of of dansylglycine with Tau-NHBr; (c) Reaction of dansylglycine with Tau-NHBr and the effect of methionine; (d) Effect of methionine on the pseudo-first-order reaction rate of dansylglycine with methionine; (d) Effect of methionine on the pseudo-first-order reaction rate of dansylglycine with Tau-NHBr. The reaction mixture was composed of 25 µ M dansylglycine, 500 µ M Tau-NHBr and Tau-NHBr. The reaction mixture wasacids composed of 25 µM dansylglycine, 500 µM Tau-NHBr and increasing concentrations of amino in 50 mM phosphate buffer, pH 7.0. The stopped-flow increasing of amino acids innm 50 LED; mM phosphate buffer, pHcut-off 7.0. The stopped-flow system systemconcentrations was set as follows: excitation, 360 and emission, 475 nm filter. was set as follows: excitation, 360 nm LED; and emission, 475 nm cut-off filter. 2.6. Comparison between Tau-NHCl and Tau-NHBr

2.6. Comparison between Tau-NHClabove, and Tau-NHBr As we have demonstrated 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, As we have demonstrated above, Tau-NHCl was unreactive with all studied compounds in this it does not mean that Tau-NHCl is completely devoid of oxidant capacity, as is confirmed, for work; which is, indeed, in agreement with its well-established poor oxidant capacity [32]. Obviously, instance, by its capacity to oxidize sulfhydryl residues in proteins [27]. Tau-NHCl is also able to it does not mean that Tau-NHCl is completely devoid of oxidant capacity, as is confirmed, for oxidize 3,3',5,5'-tetramethylbenzidine (TMB) in acid medium, the chromogenic substrate used for instance, by its capacity to oxidize activity sulfhydryl residues in proteins [27]. Tau-NHCl is also in able to determination of the chlorination of MPO [44]. Thus, aiming to quantify the difference oxidize 3,3’,5,5’-tetramethylbenzidine (TMB) in acid medium, the chromogenic substrate used reactivity between Tau-NHBr and Tau-NHCl, we used TMB as the target. It must also be noted that for determination ofacidic the chlorination activity of MPO [44]. quantify difference in instead of an pH, the reaction was conducted at pHThus, 7.0, as aiming was usedtofor the otherthe compound studiesbetween in this work. It is important to emphasize point, reactivity is significantly reactivity Tau-NHBr and Tau-NHCl, we this used TMBbecause as theits target. It must also be noted at of neutral pH, which is, reaction indeed, the for theatapplication acidic medium forother that lower instead an acidic pH, the wasreason conducted pH 7.0, asof was used for the determination of Tau-NHCl activity using TMB [44]. The results depicted in Figure 7 confirmed our compound studies in this work. It is important to emphasize this point, because its reactivity is expectation, because, at pH 7.0, Tau-NHCl was able to oxidize TMB. Its oxidant efficacy was significantly lower at neutral pH, which is, indeed, the reason for the application of acidic medium for measured (k2 = 3.1 M−1s−1) and, corroborant with the previous results, was about 100-fold lower than determination of Tau-NHCl activity using TMB [44]. The results depicted in Figure 7 confirmed our Tau-NHBr (k2 = 6.4 × 102 M−1s−1). The comparison was also made using the antioxidant curcumin, an

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 ˆ 102 M´1 s´1 ). The comparison was also made using the antioxidant curcumin, an oxidizable

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´1 and 38.2 M´1 s´1 for Tau-NHCl and polyphenol, aspolyphenol, the target. as The constants 7.3 were M´1 s7.3 oxidizable therate target. The rate were constants M −1s−1 and 38.2 M−1s−1 for Tau-NHCl Biomolecules 2016, 6, 23 8 of 17 Tau-NHBr, respectively (Figure 7). and Tau-NHBr, respectively (Figure 7).

Fluorescence (a.u.) Fluorescence (a.u.)

oxidizable polyphenol, as the target. The rate constants were 7.3 M −1s−1 and 38.2 M−1s−1 for Tau-NHCl a a) HSA 10 M and Tau-NHBr, respectively (Figure100 7). a

80

a)

100 60

da

80 40 60 20 40 0 20

c

bd c 350 400 450 b Wavelength (nm)

b)0

500

350 400 450 a Wavelength (nm)

50 Fluorescence (a.u.) Fluorescence (a.u.)

+ Tau-NHBr 200 M + HOBr 200 M +HSA 10 HOCl M 200 M + Tau-NHBr 200 M + HOBr 200 M + HOCl 200 M

b c da b c d

40

500

d

b)

50 30

ca b d

20 40 10 30

c b

20 0 450 10

500 Wavelength (nm)

550

0

Figure 6. Oxidation of HSA by HOBr, HOCl and Tau-NHBr. (a) Reaction mixtures were composed of

450 500 550 Figure 6. Oxidation of HSA by HOBr, HOCl and Tau-NHBr. (a) Reaction mixtures were composed (nm) 10 µ M HSA and 200 µ M oxidants in 5.0 mMWavelength phosphate buffer, pH 7.0. After five minutes, 250 mM of 10 µM HSA and 200 µM oxidants in 5.0 mM phosphate buffer, pH 7.0. After five minutes, 250 mM methionine was added and the HSA intrinsic fluorescence was at an excitation of 295 nm; Figure 6. Oxidation of HSA by HOBr, HOCl and Tau-NHBr. (a) measured Reaction mixtures were composed of methionine was added and the HSA intrinsic fluorescence was measured at an excitation of 295 nm; (b) Addition of 5 µ M dansylglycine to the oxidized protein and its fluorescence obtained by250 energy 10 µ M HSA and 200 µ M oxidants in 5.0 mM phosphate buffer, pH 7.0. After five minutes, mM (b) Addition of 5 tryptophan µM dansylglycine to the oxidized protein and its fluorescence obtained by energy transfer from residues. methionine was added and the HSA intrinsic fluorescence was measured at an excitation of 295 nm; transfer from tryptophan residues.

(b) Addition of 5 µ M dansylglycine to the oxidized protein and its fluorescence obtained by energy b) a) tryptophan residues. transfer from

0.2 0.3

0.003

k2 = 6.4x10 2M-1s-1 r2 = 0.9956

0.003 0.002

b) -1 kobs (s-1k) obs (s )

a) 0.3 0.4

k2 = 6.4x10 2M-1s-1 r2 = 0.9956

0.1 0.2 0.0 0.1 100

200

0.0 c) 100 0.012

200

0.010 c) 0.012 0.008 0.010 0.006 0.008 0.004 0.006 0 0.004

300 400 500 Tau-NHBr (M)

300 400 500 Tau-NHBr k2 = 38.2 M-1s-1 (M) r2 = 0.9946 k2 = 38.2 M-1s-1 r2 = 0.9946

50

100 150 200 Tau-NHBr (M)

600

k2 = 3.1 M-1 s-1 r2 = 0.9853

0.001 0.000 200

d)

250

k2 = 3.1 M-1 s-1 r2 = 0.9853

0.002 0.001

d) 0.000 600 0.003 -1 kobs (s-1k)obs (s )

-1 kobs (s-1 )kobs (s )

-1 kobs (s-1k) obs (s )

0.4

0.003 0.002

400 600 Tau-NHCl (mM)

800

200 400 600 -1 -1 k2 = 7.3 M s Tau-NHCl (mM) 2 r = 0.9712

800

k2 = 7.3 M-1s-1 r2 = 0.9712

0.002 0.001 0.001 0.000 0

100 200 300 Tau-NHCl (M)

400

0.000 0 50 100 150 200 250 0 100 200 and 300curcumin. 400 (a,b) Figure 7. Tau-NHBr versus TauNHCl as oxidants of tetramethylbenzidine Tau-NHBr (M) Tau-NHCl (M) Determination of bimolecular rate constant (k2) for the reaction between TMB and Tau-NHBr or TauNHCl. reaction mixture was composed of 1.4 of mM TMB and increasing of Figure The 7. Tau-NHBr versus TauNHCl as oxidants tetramethylbenzidine andconcentrations curcumin. (a,b) Figure 7. Tau-NHBr versus TauNHCl as oxidants of tetramethylbenzidine and curcumin. haloamines in 50 mM phosphate buffer, pH 7.0; (c,d) Determination of the bimolecular rate constants Determination of bimolecular rate constant (k2) for the reaction between TMB and Tau-NHBr or Tau(a,b) (k Determination of bimolecular rate constant (k2 ) for theTau-NHCl. reaction between TMBmixture and Tau-NHBr 2) for the curcumin and Tau-NHBr The reaction was NHCl. The reactions reaction between mixture was composed of 1.4 mMorTMB and increasing concentrations of or Tau-NHCl. The reaction mixture was composed of 1.4 mM of TMB and increasing concentrations of composed of 10 µ M curcumin and increasing concentrations haloamines in 50 mM phosphate haloamines in 50 mM phosphate buffer, pH 7.0; (c,d) Determination of the bimolecular rate constants haloamines in 50 mM phosphate buffer, pH 7.0; (c,d) Determination of the bimolecular rate constants buffer, pH 7.0. (k2) for the reactions between curcumin and Tau-NHBr or Tau-NHCl. The reaction mixture was

(k2 ) for the reactions between curcumin and Tau-NHBr or Tau-NHCl. The mixture was composed of 10 µ M curcumin and increasing concentrations of haloamines in reaction 50 mM phosphate composed of 10 µM curcumin and increasing concentrations of haloamines in 50 mM phosphate buffer, buffer, pH 7.0. pH 7.0.

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2.7. pH Effect on Tau-NHBr Reactivity In aqueous solutions, Biomolecules 2016, 6, 23

chloramine and bromamine are in equilibrium with their dichloramine 9 of 17 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 2.7. pH Effect on Tau-NHBr Reactivity of Tau-NHCl to Tau-NHCl2 is pH dependent, being favored at taurine, respectively. The conversion acidic [32]. Here, we found the same tendency for Tau-NHBr, which was progressively, converted InpH aqueous solutions, chloramine and bromamine are in equilibrium with their dichloramine and to Tau-NBr2 asforms the pH decreased. results depicted in Figure 8a results show the absorbance increase at dibromamine [32,33]. These The dihalogenated structures are the of the disproportionation 241 and 346 andtwo decrease at 288ofnm, which provides evidence for the formation of Tau-NBr 2 [33]. reaction, by nm which molecules Tau-NHX are converted to Tau-NX respectively. 2 and taurine, The conversion chemical equation below to shows the equilibrium between the monoand dihalogenated forms as The of Tau-NHCl Tau-NHCl being favored at acidic pH [32]. Here, 2 is pH dependent, wellfound as thethe pHsame dependence (Equation 2). we tendency for Tau-NHBr, which was progressively, converted to Tau-NBr2 as the + + and 346 nm pH decreased. The results depicted in Figure 8a show the absorbance increase at 241 2 HO 3 SCH2 CH2 NHBr + H ↔ HO3 SCH2 CH2 NBr2 + HO3 SCH2 CH2 NH3 (2) and decrease at 288 nm, which provides evidence for the formation of Tau-NBr [33]. The chemical 2 (Tau-NHBr) (Tau-NBr2 ) (Taurine), equation below shows the equilibrium between the mono- and dihalogenated forms as well as the pH Next, we studied the dependence (Equation 2). effect of pH on the reactivity of Tau-NHBr. Figure 8 shows the k2 obtained for the reactions between dansylglycine and Tau-NHBr at pH 5.0 and 9.0. The rate constants were (pH 5.0) 1.12 ×HO 1034SCH M−1s2−1CH , (pH 7.0) 9.5 10`1 Ø M−1s−1HO and (pH2 CH 9.0)2 NBr 1.7 ×2 101`HO M−1s3−1SCH . The2 CH pH2dependence can ` ×H NH` 2 NHBr 3 SCH 3 (2) be explained by taking account the higher reactivity forms, as has been pTauinto ´ NHBrq pTau ´ NBr2 q of the dihalogenated pTaurineq, demonstrated for Tau-NCl2 [45].

a) Absorbance

0.3 pH 4.3 pH 5.0 pH 6.0 pH 7.0 pH 8.0 pH 9.0

0.2

0.1

0.0 250 300 350 400 450 500 Wavelength (nm)

b) 6

kobs (s -1)

5

k2 = 1.10 x 10 4 M-1s-1 r2 = 0.9907

4 3 2 1 200

300 400 Tau-NHBr (M)

500

c) 0.008

kobs (s -1)

k2 = 1.7 x 10 1 M-1 s-1 2 0.006 r = 0.9942 0.004 0.002 0.000 200

300 400 Tau-NHBr (M)

500

Figure 8. 8. pH Tau-NHBr (5 mM) waswas prepared in water using a 100Figure pHeffect effecton onTau-NHBr Tau-NHBrreactivity. reactivity.(a)(a) Tau-NHBr (5 mM) prepared in water using a fold excess of taurine. The solution was diluted to 0.25 mM of NaH 2PO4/Na2HPO4 (50 mM) solutions 100-fold excess of taurine. The solution was diluted to 0.25 mM of NaH2 PO4 /Na2 HPO4 (50 mM) combinedcombined to reach the (b,c) Reactivity Tau-NHBr with dansylglycine at pH 5.0 and 9.0. solutions to indicated reach the pH; indicated pH; (b,c) of Reactivity of Tau-NHBr with dansylglycine at pH 5.0 and 9.0.

2.8. Reactivity of Tau-NHBr with Hydrogen Peroxide and Formation of Singlet Oxygen

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-NHBr at pH 5.0 and 9.0. The rate constants were (pH 5.0)

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 2O2 [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 emphasize2016, that the extremely low light emission is due to the inadequacy of a conventional Biomolecules 6, 23 10 of 17 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 1.1 ˆ 104 M´1 s´1 , (pH 7.0) 9.5 ˆ 101 M´1 s´1 and (pH 9.0) 1.7 ˆ 101 M´1 s´1 . The pH dependence results depicted in Figure 10b show that the light emission increased almost two orders of magnitude can be explained by taking into account the higher reactivity of the dihalogenated forms, as has been compared to the reaction without melatonin. The light emission is dependent on both H 2O2 and demonstrated for Tau-NCl2 [45]. melatonin, which excluded the possibility of direct interaction between melatonin and Tau-NHBr or melatonin andofHTau-NHBr 2O2 as a source of the light emission. Thus, the increase in emission is consistent with 2.8. Reactivity with Hydrogen Peroxide and Formation of Singlet Oxygen the cleavage of the pyrrole ring of melatonin and formation of N-acetyl-N-formyl-5Another property that distinguishes Tau-NHCl from Tau-NHBr isbeen the capacity of the between latter to methoxykynuramine (AFMK), a chemiluminescent reaction [47] that has demonstrated ´ 1 ´ 1 react with H [33]. Here, this[48]. chemical propertywith was this confirmed andthe theformation k2 (3.9 M ofsAFMK ) obtained by 2 O2singlet melatonin and oxygen In accordance proposal, was also measuring the decay of Tau-NHBr as a function of increasing concentrations of H O (Figure 9). 2 2 obtained here (Figure 10c).

a) H202 1.25 mM 2.50

Absorbance

288 nm

0.10 0.08

3.75

0.06

5.00

0.04

7.50

0.02 0.00 0

100

b)

200 Time (s)

300

400

6

8

0.04 k2 = 3.9 M-1s-1 r2 = 0.9944

kobs (s-1)

0.03 0.02 0.01 0.00 0

2

4 H2O2 (mM)

Figure 9. 9. Reactivity with H H2O O2. (a) Kinetic profile of Tau-NHBr consumption for Figure Reactivity of of Tau-NHBr Tau-NHBr with 2 2 . (a) Kinetic profile of Tau-NHBr consumption for determination of kobs under under pseudo-first-order pseudo-first-order experimental experimental conditions. conditions. The reaction mixture mixture was was determination of kobs The reaction composed of 250 µ M Tau-NHBr and increasing concentrations of H 2O2 in 50 mM phosphate buffer, composed of 250 µM Tau-NHBr and increasing concentrations of H2 O2 in 50 mM phosphate buffer, pH 7.0; rate constant (k2)(kfor the reaction between H2O2 and TaupH 7.0; (b) (b) Determination Determinationofofthe thebimolecular bimolecular rate constant 2 ) for the reaction between H2 O2 and NHBr. Tau-NHBr.

2.9. Reactivity of Tau-NHBr with Linoleic Acid Activated eosinophils are an important in vivo source of singlet oxygen. The formation of this Among the biomolecules susceptible the deleterious effects reactive oxygen electronically excited form of oxygen is due totothe reaction between HOBrprovoked and H2 O2by [46]. We found that species (ROS), unsaturated fattyas acids position. electrophilic reagents, HOCl Tau-NHBr retains this capacity, can occupy be seenan byimportant the ultra-weak lightAs emission produced during the and HOBr are well established as inducers of the formation of halohydrin by reacting with the double reaction course originating from the decay of singlet oxygen (Figure 10a). It is important to emphasize bonds mono- and polyunsaturated [49,50]. For thisofreason, we also investigated that theinextremely low light emission fatty is dueacids to the inadequacy a conventional luminometerthe to reactivitythe ofemission Tau-NHBr with linoleic asemits a model polyunsaturated measure of singlet oxygen, acid which in theofinfrared region (1200fatty nm).acids. Hence,Intothese 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 H2 O2 and melatonin, which excluded the possibility of direct interaction between melatonin and Tau-NHBr or melatonin and H2 O2 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

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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 Biomolecules 2016, 6, 23 11 of 17 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. could be expected from the previous results, Tau-NHCl still [48]. less chemiluminescent reactionAs [47] that has been demonstrated between melatonin and singletwas oxygen reactive. In accordance with this proposal, the formation of AFMK was also obtained here (Figure 10c).

a) Light Emission (a.u.)

150

Tau-NHBr + H2O2 Tau-NHBr H2O2

100

50

0 0

100 200 Time (s)

300

b) Light Emission (a.u.)

8000 Complete system - H2O2 - MLT - Tau-NHBr

6000 4000 2000 0 0

50 Time (s)

100

c) AFMK

Fluorescence (a.u.)

0.15

Complete system - H2O2

0.10 0.05 0.00 -0.05 0

5

10 Time (min)

15

Figure 10. Evidence Evidence of ofoxygen oxygensinglet singletproduction. production.(a)(a) Intrinsic light emission by the reaction of 0.25 Figure 10. Intrinsic light emission by the reaction of 0.25 mM mM Tau-NHBr H 250 O 2 mM in 50 mM phosphate buffer, pH 7.0; (b) Melatonin-mediated light Tau-NHBr with 5with mM5HmM O in phosphate buffer, pH 7.0; (b) Melatonin-mediated light emission 2 2 emission by the consisting reaction consisting of 0.25 mM Tau-NHBr with H2O2 and 1 mM melatonin in by the reaction of 0.25 mM Tau-NHBr with 5 mM H25OmM 2 and 1 mM melatonin in 50 mM 50 mM phosphate buffer, pH 7.0 (complete system); (c) HPLC analysis of the reaction and phosphate buffer, pH 7.0 (complete system); (c) HPLC analysis of the reaction mixture andmixture evidence of evidence of theofformation of AFMK (peak at 6 min) by comparison with a pure AFMK. the formation AFMK (peak at 6 min) obtained by obtained comparison with a pure AFMK.

2.9. 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

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of incubation, Tau-NHBr reacted slowly, with only 50% consumed in 60 min of incubation. As could 12 of 17 previous results, Tau-NHCl was still less reactive. 12 of 17 Consumedoxidant M) oxidant( (M) Consumed

Biomolecules 2016, 6, 23the be expected from Biomolecules 2016, 6, 23

Tau-NHBr Tau-NHBr HOBr HOBr Tau-NHCl Tau-NHCl HOCl HOCl

60 60 40 40 20 20 0 00 0

20 20

40 60 40 60 Time (min) Time (min)

80 80

100 100

Figure 11. Reactivity M Tau-NHBr, Figure Reactivitywith withlinoleic linoleicacid. acid.The Thereaction reactionmixtures mixtureswere werecomposed composedof of50 50µµµM Tau-NHBr, Figure 11. 11. Reactivity with linoleic acid. The reaction mixtures were composed of 50 M Tau-NHBr, Tau-NHCl, HOBr, or HOCl and 100 µµM M linoleic acid in 50 mM phosphate buffer, pH 7.0. After the Tau-NHCl, HOBr, or HOCl and 100 phosphate Tau-NHCl, HOBr, or HOCl and 100 µ M linoleic acid in 50 mM phosphate buffer, pH 7.0. After After the the indicated time, the remaining oxidants were measured by the TNB method. The results are the mean indicated time, the remaining oxidants were measured by the TNB method. The results are the mean indicated time, the remaining oxidants were measured by the TNB method. The results are the mean of of three three experiments. experiments. HOCl HOCl and and HOBr HOBr were were totally totally consumed consumed in in the the first first minute of reaction. of three experiments. HOCl and HOBr were totally consumed in the first minute of reaction.

2.10. 2.10. Reactivity of of Tau-NHBr Tau-NHBr with with Nucleosides 2.10. Reactivity Reactivity of Tau-NHBr with Nucleosides Nucleosides Other Other well established endogenous targets targets for for hypohalous hypohalous acids acids are are nucleosides, nucleosides, nucleotides, nucleotides, Other well well established established endogenous endogenous targets for hypohalous acids are nucleosides, nucleotides, DNA and RNA. For instance, 5-chlorouracil and 5-bromouracil are formed from the MPO DNA andRNA. RNA. instance, 5-chlorouracil and 5-bromouracil are formed the MPO DNA and ForFor instance, 5-chlorouracil and 5-bromouracil are formed from thefrom MPO catalyzed catalyzed oxidation of uracil, and 5-chloro-2'-deoxycytidine, 8-chloro-2'-deoxyadenosine and 8-chloro-2'catalyzed oxidation of uracil, and 5-chloro-2’-deoxycytidine, 8-chloro-2’-deoxyadenosine and 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 8-chloro-2’-deoxyguanosine from the of treatment of DNA [51]. the Recently, the deoxyguanosine result fromresult the treatment DNA with HOClwith [51].HOCl Recently, miscoding properties of 8-chloro-2'-deoxyguanosine have been demonstrated, which highlights the potential miscoding properties of 8-chloro-2’-deoxyguanosine have been demonstrated, which highlights properties of 8-chloro-2'-deoxyguanosine have been demonstrated, which highlights the potential importance of HOCl-mediated reactions pathogenesis inflammation-driven carcinogenesis the potential of HOCl-mediated reactions in theof pathogenesis of inflammation-driven importance ofimportance HOCl-mediated reactions in in the the pathogenesis of inflammation-driven carcinogenesis [52]. Similarly, the presence of 8-bromo-2'-deoxyguanosine has been proposed as a substance carcinogenesis Similarly, presence of 8-bromo-2’-deoxyguanosine has been proposed that as a [52]. Similarly, [52]. the presence ofthe 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 substance that may increase mutagenic potential at the site of inflammation [53]. For this reason, we 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 also evaluated the reactivity of Tau-NHBr with nucleosides. However, the superposition of absorption the reactivity of Tau-NHBr with nucleosides. However, the superposition of absorption spectra between Tau-NHBr, and nucleosides impeded the direct measurement of their spectra Tau-NHBr, and the nucleosides impeded direct measurement theirconsumption. consumption. betweenbetween Tau-NHBr, and the the nucleosides impeded the the direct measurement ofof their consumption. Hence, we again employed an indirect experimental approach by incubating the nucleosides Hence, we again employed an indirect experimental approach by incubating the nucleosides with Hence, we again employed an indirect experimental approach by incubating the nucleosides with with the oxidant and then measuring the remaining oxidant with the TNB reagent. The results depicted the oxidant and and then then measuring measuring the the remaining remaining oxidant oxidant with TNB reagent. reagent. The The results results depicted depicted in in the oxidant with the the TNB in Figure 12 show that, in contrast to HOBr, Tau-NHBr was significantly less reactive with nucleosides. Figure 12 show that, in contrast to HOBr, Tau-NHBr was significantly less reactive with nucleosides. Figure 12 show that, in contrast to HOBr, Tau-NHBr was significantly less reactive with nucleosides. URIDINE URIDINE ADENOSINE ADENOSINE THYMIDINE THYMIDINE GUANOSINE GUANOSINE CYTIDINE CYTIDINE URIDINE URIDINE ADENOSINE ADENOSINE THYMIDINE THYMIDINE GUANOSINE GUANOSINE CYTIDINE CYTIDINE

HOBr HOBr

Tau-NHBr Tau-NHBr 0 50 100 0 50 100 Consumed HOBr or Tau-NHBr (M) Consumed HOBr or Tau-NHBr (M)

Figure 12. Reactivity of Tau-NHBr and HOBr with nucleosides. The reaction mixtures were composed Figure 12. Reactivity Tau-NHBr and HOBr with The reaction were composed Figure Reactivity of of with nucleosides. nucleosides. Thephosphate reaction mixtures mixtures were of 100 µ12. M Tau-NHBr orTau-NHBr HOBr andand 100 HOBr µ M nucleosides in 50 mM buffer, pH 7.0.composed After 30 of 100 µ M Tau-NHBr or HOBr and 100 µ M nucleosides in 50 mM phosphate buffer, pH 7.0. After 30 of 100 Tau-NHBr or HOBr and 100 µM by nucleosides in 50 mMThe phosphate buffer, pH 7.0. After min theµM remaining oxidants were measured the TNB method. results are the mean of three min the remaining oxidants were measured by the TNB method. The results are the mean of three 30 min the remaining oxidants were measured by the TNB method. The results are the mean of experiments. experiments. three experiments.

3. 3. Experimental Experimental Section Section 3.1. Chemicals and 3.1. Chemicals and Solutions Solutions Taurine, Taurine, tryptophan, tryptophan, tyrosine, tyrosine, methionine, methionine, cysteine, cysteine, melatonin, melatonin, serotonin, serotonin, N-acetyl-N-formyl-5N-acetyl-N-formyl-5methoxykynuramine (AFMK), TMB, 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB), methoxykynuramine (AFMK), TMB, 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB), dansylglycine, dansylglycine, linoleic linoleic acid, acid, salicylic salicylic acid, acid, curcumin, curcumin, adenosine, adenosine, cytidine, cytidine, guanosine, guanosine, thymidine, thymidine, uridine uridine and and HSA HSA

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3. Experimental Section 3.1. Chemicals and Solutions Taurine, tryptophan, tyrosine, methionine, cysteine, melatonin, serotonin, N-acetyl-N-formyl-5methoxykynuramine (AFMK), TMB, 5,5’-dithiobis-(2-nitrobenzoic acid) (DTNB), dansylglycine, linoleic acid, salicylic acid, curcumin, adenosine, cytidine, guanosine, thymidine, uridine and HSA were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). The chemicals used for preparation of phosphate buffers and others solutions were of analytical grade. HOCl was prepared by diluting a 5% stock solution and the concentration was determined spectrophotometrically after dilution in 0.01 M NaOH, pH 12 (ε292nm = 350 M´1 cm´1 ). HOBr was synthesized by combining 100 mM HOCl and 200 mM NaBr in water [33]. Tau-NHCl was prepared by the addition of 5 mM HOCl to 50 mM taurine in 50 mM sodium phosphate buffer at pH 7.0 and its concentration was determined spectrophotometrically (ε252nm = 429 M´1 cm´1 ) [25]. Tau-NHBr was prepared by the addition of 5 mM HOBr to 500 mM taurine in 50 mM sodium phosphate buffer at pH 7.0 and its concentration was determined spectrophotometrically (ε288nm = 430 M´1 cm´1 ) [25]. TNB solution was prepared by dissolving 2 mM DTNB in 50 mM phosphate buffer, pH 7.0. Then, this solution was titrated to pH 12 with 0.1 M NaOH to promote its hydrolysis, and after 5 min the pH was brought back to 7.4 with hydrochloric acid. Then, its concentration was determined by its absorbance (ε412nm = 14,100 M´1 cm´1 ) [54]. A Perkin Elmer Lambda 35 UV-visible spectrophotometer (Manufacturer, Shelton, CT, USA) was used for the UV-Vis measurements. 3.2. 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 (kobs ) was obtained by fitting the fluorescence or absorbance decay of the studied compound to a single exponential decay equation, as follows (Equation 3): S “ S0 ˆ e´kobs ˆ t ,

(3)

where: S is the fluorescence or absorbance as a function of time S0 is the initial fluorescence or absorbance From the kobs values obtained at various concentrations, the bimolecular rate constants (k2 ) were calculated from the slope of the linear regression as follows (Equation 4): reaction rate “ k2 ˆ rAs ˆ rBs,

(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 (k2 ) was calculated (Equation 5). If rAs " rBs, then, reaction rate “ kobs ˆ rBs Then, kobs “ k2 ˆ rAs,

(5)

The k2 is the slope of the linear fit of kobs 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

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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. 3.3. 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. 3.4. Reactions with 3,3’,5,5’-tetramethylbenzidine (TMB), Curcumin, Hydrogen Peroxide and Linoleic Acid 3,3’,5,5’-tetramethylbenzidine: The reaction medium was composed of 1.4 mM TMB and increasing concentrations of haloamines in 50 mM phosphate buffer, pH 7.0. The reaction was monitored at 650 nm using conventional spectrophotometry. 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 H2 O2 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 (250 ˆ 4.6 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 (ε412nm = 14,100 M´1 cm´1 ). 4. 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. Acknowledgments: This work was supported by grants (2013/08784-0 and 2105/08267-1) from the São Paulo Research Foundation (FAPESP), Coordination for Enhancement of Higher Education Personnel (CAPES) and National Council for Scientific and Technological Development (CNPq, 440503/2014-0).

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Author Contributions: Valdecir Farias Ximenes and Luiza De Carvalho Bertozo conceived and designed the experiments; Valdecir Farias Ximenes and Luiza De Carvalho Bertozo performed the experiments; Valdecir Farias Ximenes, Aguinaldo Robinson De Souza, Nelson Henrique Morgon and Luiza de Carvalho Bertozo analyzed the data; Valdecir Farias Ximenes wrote the paper. Conflicts of Interest: The authors declare no conflict of interest.

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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 ap...
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