Sulfite salts are widely used as antibrowning agents in food processing. Nevertheless, the exact mechanism by which sulfite prevents enzymatic browning has remained unknown. Here, we show that sodium hydrogen sulfite (NaHSO3) irreversibly blocks the active site of tyrosinase from the edible mushroom Agaricus bisporus, and that the competitive inhibitors tropolone and kojic acid protect the enzyme from NaHSO3 inactivation. LC-MS analysis of pepsin digests of NaHSO3-treated tyrosinase revealed two peptides showing a neutral loss corresponding to the mass of SO3 upon MS2 fragmentation. These peptides were found to be homologous peptides containing two of the three histidine residues that form the copper-B-binding site of mushroom tyrosinase isoform PPO3 and mushroom tyrosinase isoform PPO4, which were both present in the tyrosinase preparation used. Peptides showing this neutral loss behavior were not found in the untreated control. Comparison of the effects of NaHSO3 on apo-tyrosinase and holo-tyrosinase indicated that inactivation is facilitated by the active site copper ions. These data provide compelling evidence that inactivation of mushroom tyrosinase by NaHSO3 occurs through covalent modification of a single amino-acid residue, probably via addition of HSO3− to one of the copper-coordinating histidines in the copper-B site of the enzyme.
Tyrosinase is a polyphenol oxidase (PPO) that catalyzes either the o-hydroxylation and subsequent oxidation of phenolic compounds, or the oxidation of o-diphenolic compounds. The resulting o-quinones, in turn, polymerize into brown pigments . In foods, this reaction is often unwanted, and accounts for major losses in fruit and vegetable processing. Among the possible ways to inhibit enzymatic browning during food processing, sulfite salts are widely used as additives. When dissolved, these sulfite salts yield a mixture of SO32− and HSO3−, the ratio of which depends on the pH. Despite the importance of sulfite as an antibrowning agent, the exact mechanism by which it prevents enzymatic browning has remained unknown.
Recently, we demonstrated that sulfite inhibits browning by trapping enzymatically formed o-quinones in colorless addition products, referred to as sulfo-phenolics. This was demonstrated both when phenolics were extracted from potato in the presence of sodium hydrogen sulfite (also known as sodium bisulfite, NaHSO3) , and in model systems consisting of commercially available tyrosinase from the edible mushroom Agaricus bisporus (EC 184.108.40.206) and chlorogenic acid . Furthermore, it was found  that the catalytic activity of mushroom tyrosinase was inhibited by NaHSO3 in a relatively slow, time-dependent way.
Here, we further investigated the time-dependent inhibition of mushroom tyrosinase by sulfite. We hypothesized that covalent modification of the enzyme by HSO3− might be responsible for the observed time-dependent inhibition, and that the active site would be the obvious target for such a modification.
The catalytic center of mushroom tyrosinase, a type-3 copper protein, contains two copper ions, each of which is coordinated by three histidine residues. The two copper-binding sites, the copper-A site and the copper-B site, are highly conserved among different species . The copper center can be in different states, depending on the oxidation state of the copper ions and their interaction with oxygen . In the met state, the copper ions are in the Cu(II) oxidation state, with hydroxyl ligands bridging the two copper ions. In this state, an o-diphenol can bind to the copper ions, after which oxidation takes place and an o-quinone is released, reducing Cu(II) to Cu(I). The resulting deoxy state is subsequently oxidized to the Cu(II) oxy state by binding oxygen. Oxy tyrosinase can bind either an o-diphenol that is oxidized, or a monophenol, which is o-hydroxylated and subsequently oxidized, after which the cycle described can take place again. In the resting state, tyrosinase is considered to be predominantly in the met state, causing a lag phase to be observed when the activity of tyrosinase is assayed with monophenolic substrates.
One of the histidines (His85) coordinating the copper-A ion in mushroom tyrosinase is covalently linked via a thioether to Cys83, which fixes the orientation of this histidine side chain . This thioether is also found in PPOs from other species, e.g. grape (Vitis vinifera) , sweet potato (Ipomoea batatas) , and the fungus Neurospora crassa . It has been proposed that thioether formation is catalyzed by presence of copper in the active site . The copper-catalyzed formation of the thioether might indicate that the other histidine residues in the active site of mushroom tyrosinase are also reactive with sulfur-containing nucleophiles such as HSO3−.
To elucidate its mechanism, the time-dependent inhibition of mushroom tyrosinase by NaHSO3 was investigated in more detail. First, the effects of sulfite on the monophenolase and diphenolase activity of tyrosinase were compared. Next, to further characterize the molecular basis of tyrosinase inhibition by sulfite, we investigated the reversibility of inhibition and the possible covalent modification of amino-acid residues in the active site.
Inhibition of the monophenolase versus the diphenolase reaction
In order to determine whether NaHSO3 selectively inhibits either the monophenolase or the diphenolase reaction catalyzed by tyrosinase, oxygen consumption measurements were performed with combinations of l-tyrosine, l-3,4-dihydroxyphenylalanine (l-Dopa), NaHSO3, and tyrosinase (Fig. 1A). When l-Dopa was incubated with tyrosinase in the presence of NaHSO3, oxygen consumption was initially similar to that in a control incubation of l-Dopa and tyrosinase. However, in time, a leveling off of oxygen consumption occurred, until a plateau was reached before all oxygen was consumed. This observation is in accordance with our previous observations on the time-dependent inhibition of tyrosinase-catalyzed oxidation of chlorogenic acid by NaHSO3 . With tyrosinase and tyrosine, an initial lag phase (~ 200 s) before a linear decrease in oxygen concentration was observed. The lag phase is typical for the activity of tyrosinase on monophenolic substrates . When the combination of tyrosine, NaHSO3 and tyrosinase was incubated, no oxygen consumption was observed, also not after a time corresponding to the previously described lag phase. On the basis of this observation, one might conclude that NaHSO3 inhibited the monophenolase reaction more strongly than the diphenolase reaction. Alternatively, the total inhibition of oxygen consumption might also be explained by the typical lag phase for the oxidation of monophenols combined with the time-dependent inhibition of tyrosinase by NaHSO3: if the time required to inhibit tyrosinase by NaHSO3 coincides with the lag phase of tyrosinase activity on tyrosine, no oxygen consumption will be observed. To investigate whether this was indeed the case, an experiment was carried out in which NaHSO3 was added to an incubation of tyrosine and tyrosinase 300 s after the incubation started, when the oxidation of tyrosine was occurring at its linear rate. It was observed that NaHSO3 showed a similar time-dependent effect as with the oxidation of l-Dopa: initially, the reaction rate was similar to that of the control without NaHSO3, but in time it leveled off until a plateau was reached. On the basis of this experiment, it was concluded that the time-dependent inhibition of tyrosinase by NaHSO3 is independent of the type of reaction, monophenolase or diphenolase, that it catalyzes.
Tyrosinase is irreversibly inactivated by NaHSO3
To establish whether the observed time-dependent inhibition of tyrosinase by NaHSO3 is reversible, a preincubation and reactivation experiment was performed. When tyrosinase was incubated with NaHSO3, which was subsequently removed, the enzyme, upon incubation with l-Dopa, showed decreased oxygen consumption as compared with a diafiltered, CuCl2-treated control (Fig. 1B). To investigate whether the inactivation of tyrosinase by NaHSO3 involves the loss of copper ions, reactivation was attempted by supplementing pretreated tyrosinase with CuCl2. As a control, an experiment with dithiothreitol, which is known to reversibly inhibit tyrosinase , was performed. When the activities of these pretreated tyrosinase samples were compared (Fig. 1B), it was found that copper supplementation did not reactivate NaHSO3-inactivated tyrosinase, whereas it did reactivate dithiothreitol-inactivated tyrosinase. On the basis of these results, it was concluded that NaHSO3 irreversibly inactivates tyrosinase, and that the inactivation is not caused by chelation of copper ions.
Tyrosinase-mediated sulfo-Dopa formation
During the time-dependent inhibition of tyrosinase-catalyzed oxidation of l-Dopa with NaHSO3, no color formation was observed (data not shown). Although tyrosinase was active, as indicated by oxygen consumption (Fig. 1A), no dopachrome was formed from the resulting Dopa-quinone. This might be explained by the formation of sulfonated derivatives of l-Dopa, trapping the o-quinones and making them unavailable for further reactions. To investigate whether the sulfonation of Dopa-quinone did indeed occur, reaction products of incubations of l-Dopa with tyrosinase, with and without NaHSO3, were analyzed by RP-UHPLC-PDA-MS (Fig. 1C–E; Table 1). After incubation of l-Dopa with tyrosinase, two peaks were observed. Peak 1 was identified as residual l-Dopa, both by MS and by comparison with the untreated l-Dopa sample. Peak 2 showed a molecular mass of 193 Da, corresponding to the mass of dopachrome. Furthermore, it also had an absorption maximum at 475 nm, corresponding to the characteristic red color of dopachrome .
Table 1. Peak annotation of RP-UHPLC-PDA-MS analysis of the reaction products of l-Dopa with NaHSO3 and tyrosinase
When l-Dopa was incubated with tyrosinase in the presence of NaHSO3, three new peaks were observed in the UV280 nm trace, besides residual l-Dopa. Compounds 3, 4 and 5 all had a mass of 277 Da, which corresponds to the mass of l-Dopa with an additional 80 Da. This mass can be explained by the addition of HSO3− to l-Dopa. Assuming a similar reaction as observed earlier for the sulfonation of chlorogenic acid , we suggest that these three reaction products represent l-Dopa substituted with HSO3− at the three free positions of the phenyl ring. Considering steric influences of the phenyl ring substituents, and based on data on the substitution of dihydrocaffeic acid and catechin with glutathione [13, 14], the most abundant sulfo-Dopa isomer probably represents l-Dopa substituted at the 5-position on the phenyl, followed in abundance by l-Dopa substituted at the 6-position and at the 2-position (corresponding to peaks 4, 3 and 5, respectively).
Competitive inhibitors hinder NaHSO3-mediated inactivation of tyrosinase
To investigate the possible location of action of NaHSO3 in its inactivation of tyrosinase, tyrosinase pretreatment with NaHSO3 was performed in the presence of competitive tyrosinase inhibitors. If NaHSO3 could not inactivate tyrosinase when its active site was occupied by an inhibitor, it would be an indication that NaHSO3 needs access to the active site to exert its action. The competitive inhibitors kojic acid and tropolone [15, 16] were used for this experiment.
Pretreatment with NaHSO3 led to partial or complete inactivation of tyrosinase, depending on the concentration of NaHSO3 used (Fig. 2). Pretreatment of tyrosinase with tropolone and kojic acid did not lead to inactivation after their removal by diafiltration, confirming that inhibition by these compounds is indeed reversible. When tyrosinase was incubated with equimolar amounts of NaHSO3 and tropolone, no inactivation of tyrosinase was observed. When the molar ratio of NaHSO3 to tropolone was increased to 10 : 1, partial inactivation of tyrosinase as compared with the control was observed (Fig. 2A).
Combined pretreatment with equimolar amounts of NaHSO3 and kojic acid resulted in similar effects as with tropolone: the enzymatic activity was not affected. In contrast to the findings with tropolone, tyrosinase was completely inactivated when the ratio of NaHSO3 to kojic acid was increased to 10 : 1 (Fig. 2B).
These observations show that the presence of competitive inhibitors in the active site of tyrosinase prevents inactivation by NaHSO3, which suggests that the NaHSO3-mediated inactivation of tyrosinase occurs in the active site. The observation that tropolone prevents inactivation at a higher NaHSO3/inhibitor ratio than that for kojic acid is probably attributable to the higher affinity of tropolone than of kojic acid for the active site [15, 16].
The copper-B site of mushroom tyrosinase is covalently modified by NaHSO3
Considering the irreversibility of tyrosinase inactivation by NaHSO3 and the fact that inactivation seems to take place in the active site, it was hypothesized that a covalent modification of active site amino acids was responsible for the inactivation.
A NaHSO3-pretreated and an untreated tyrosinase sample were digested with pepsin, and the resulting peptides were analyzed by RP-UHPLC-MSn (Fig. 3A). If a sulfonic acid group was covalently linked to a peptide, a mass increase of 80 Da as compared with an untreated control would be expected. Automated screening of MS2 data for neutral losses of 80 Da was performed. Two peaks showing a neutral loss of 80 Da (m/z 536.8 and m/z 532.8), which were not found in the untreated control, clearly stood out in the NaHSO3-treated tyrosinase (Fig. 3B). The MS2 spectra for these two peaks were atypical for peptide fragmentation, showing only one major peak, representing the parent ion minus the neutral loss of 80 Da. Fragmentation of product ions that showed a neutral loss of 80 Da was achieved by MS3 experiments performed on these product ions. As an example, the MS2 spectrum of the peak with an m/z of 532.8 is shown (Fig. 3C); the fragmentation of this yielded only one fragment, with an m/z of 492.8. Both the precursor ion and the fragment ion were doubly charged, as was clearly shown by the isotope pattern. Thus, the difference of 40 in m/z values corresponds to a mass loss of 80 Da. Fragmentation of the ion with an m/z of 492.8 resulted in a typical peptide fragmentation spectrum (Fig. 3D). De novo sequencing of the spectrum resulted in the amino acid sequence MVHNTVHF (theoretically doubly charged ion m/z 492.7), which corresponds to a region of the copper-B site of mushroom tyrosinase isoform PPO3 (amino-acid residues 257–264) (Fig. 4). Similarly, MS3 on the fragment ion with an m/z of 496.8 resulted in the amino acid sequence EAVHDDIHG (theoretically doubly charged ion m/z 496.7), which corresponds to a region of the copper-B site of mushroom tyrosinase isoform PPO4 (amino-acid residues 248–256) (Fig. 4). The peptides MVHNTVHF and EAVHDDIHG, without an extra mass of 80 Da, were also found in the MS trace of the control digest, at different retention times. The protease digestion and UHPLC-MSn analysis was repeated with chymotrypsin (LC-MS data not shown), resulting in four peptides (EMVHNTVHF and VHNTVHF of PPO3; SLEAVHDDIHGF and EAVHDDIHGF of PPO4) showing the same neutral loss behavior as the peptides found in the peptic digest, and corresponding to the same region of the copper-B site of tyrosinase (Fig. 4). Peptides showing a neutral loss corresponding to the addition of two sulfonic acid groups were not found. These results indicated that covalent addition of a single HSO3− to an active site amino acid in the copper-B site occurs in mushroom tyrosinase. Examination of the similarities between the sulfite-modified peptides found showed that the only amino-acid residues that they have in common are valine and histidine (Fig. 4). The nucleophile sulfite would need an electrophilic reaction site for attachment to an amino acid. The imidazole ring of histidine is electron-rich, making it prone to electrophilic attack. Nevertheless, nucleophilic addition on imidazole has also been reported , as well as nucleophilic addition of the sulfite ion to heteroaromatic rings . Moreover, the coordination with copper makes the imidazole side chain of histidine more electron-poor, and thus more prone to nucleophilic attack. Valine, on the other hand, has an alkyl side chain without any functional group. Considering the reactivity of the other amino-acid residues of the modified peptides, no functional groups that are prone to react with a sulfite group are present. This strongly suggests that sulfite is attached to one of the copper-B-coordinating histidines (His259 or His263 in PPO3; His251 or His255 in PPO4). To confirm that modification only occurred on one of these positions and not on the third copper-B-coordinating histidine residue, peptides containing this third histidine residue (His296 for PPO3, and His283 for PPO4) were searched for. Peptides containing His296 from PPO3 were not found for either the NaHSO3-treated sample or the control sample, whereas peptides from PPO4 containing His283 were found, in unmodified form (Fig. 4).
The copper-A site thioether of mushroom tyrosinase is not modified by sulfite
Considering the fact that a histidine residue in the copper-B site of tyrosinase appeared to be covalently modified by NaHSO3, we investigated whether the three copper-A-coordinating histidine residues in NaHSO3-treated tyrosinase were also affected by NaHSO3. Peptides containing the residues His61 and His94 of PPO3 and peptides containing the residues His57 and His91 of PPO4 were found in protease digests of both NaHSO3-treated and untreated tyrosinase (Fig. 4). The third copper-A-coordinating histidine residue in PPO3, His85, is engaged in a thioether bond with Cys83 , which will affect the molecular mass of peptides containing this thioether bond. Depending on its specificity, pepsin digestion of PPO3 would theoretically result in the peptides CTHGTVL or YKANYCTHGTVL. The theoretical molecular masses of these peptides are 729.3 and 1368.6 Da, respectively; these would be 727.3 and 1366.6 Da if the thioether is present. The latter masses were found in the LC-MS data, for both the untreated and the sulfite-treated tyrosinase samples (Fig. 5). The identity of the peptides was confirmed by MS/MS spectra. The masses of the b and y ions representing fragments of the peptide including the thioether matched the theoretical masses minus two, whereas the masses of the ions not including the thioether corresponded to their theoretical masses (Fig. 5C,D). The thioether containing peptide CTHSQVL from PPO4 was found in the same way. Similar MS evidence of a Cys-His thioether in molluskan hemocyanin, a type-3 copper protein functioning as a dioxygen carrier, has been provided previously . Taken together, these results demonstrate that the copper-A site of mushroom tyrosinase is not affected by sulfite treatment.
Copper ions facilitate NaHSO3 modification of the copper-B site of tyrosinase
To investigate whether the copper ions play a role in the covalent modification of the copper-B site of tyrosinase, apo-tyrosinase and holo-tyrosinase were incubated with NaHSO3. EDTA, a well-known metal chelator, has been previously reported to inhibit tyrosinase . Under our experimental conditions, however, no inactivation of tyrosinase by EDTA occurred (data not shown). As we found that dithiothreitol treatment resulted in reversible inactivation of tyrosinase (Fig. 1B), probably because of copper chelation , dithiothreitol was used to remove the copper ions from tyrosinase prior to NaHSO3 treatment.
On comparison of the activity of NaHSO3-pretreated holo-tyrosinase with that of NaHSO3-pretreated apo-tyrosinase, it can be seen that NaHSO3 treatment had a larger effect on active tyrosinase than on the dithiothreitol-inactivated enzyme (Fig. 6). Tyrosinase pretreated with dithiothreitol and subsequently reactivated with CuCl2 showed full recovery of activity, as compared with a control sample that was subjected to three cycles of diafiltration and CuCl2 treatment. These results suggest that the presence of copper is not essential for NaHSO3-mediated inactivation of mushroom tyrosinase, but the presence of copper facilitates the inactivation.
Although sulfite is widely used to inhibit enzymatic browning, the exact mechanism of this inhibition has remained unknown. Previously, we found that sulfite prevents the formation of brown pigments by converting o-quinones into colorless sulfo-phenolics, and by time-dependent inhibition of tyrosinase activity . Here, using different pretreatments, we demonstrated that this time-dependent inhibition is caused by irreversible inactivation of mushroom tyrosinase. The inactivation was likely to be the result of covalent modification of one of the copper-B-coordinating histidine residues, which is probably catalyzed by copper in the active site.
A possible explanation for the covalent reaction of sulfite with a histidine residue might be a sulfur lone pair-initiated nucleophilic addition of HSO3− to Cε1 of histidine, followed by hydride removal upon oxidation to restore aromaticity. As it was found that the presence of copper facilitated inactivation by NaHSO3, the interaction of Nε2 with Cu(II) probably results in reduced electron density of Cε1, in this way making it more prone to nucleophilic addition. Moreover, Cu(II) possibly promotes the oxidative step through reduction to Cu(I) (Fig. 7B). A similar mechanism has been suggested for the formation of the thioether bond in grape PPO  (Fig. 7A). Alternatively, addition of HSO3− to one of the nitrogens in the histidine side chain might occur. Reactions of sulfite with the N5 atom of flavin in different flavoproteins are known to occur, depending on the amino acids surrounding the flavin-binding site [20, 21]. A possible mechanism of HSO3− addition to Nε2 is proposed in Fig. 7C. In this mechanism, restoration of aromaticity through hydride removal upon oxidation would lead to loss of the copper-coordinating ability of Nε2. MS2 of the sulfite-substituted peptides resulted in only a single fragment with a neutral loss corresponding to the mass of SO3. The relative ease with which this fragmentation occurred might be an indication that substitution on Nε2 is more likely than on Cε1, as a C–S bond is likely to be more stable than an N–S bond. Looking at the active site cavity , it seems that His263 is more exposed than His259 (Fig. 7D). As His263 is more accessible, we speculate that it is more likely that sulfite addition takes place on this histidine residue.
We demonstrated that modification of a single amino acid in the copper-B site irreversibly inactivated tyrosinase, probably by addition to a copper-coordinating histidine residue. The reason why sulfite-modified tyrosinase is inactivated remains to be determined. If sulfonation occurs on Cε1, a possible explanation might be that the sulfite occupies too much space in the active site for substrates to bind efficiently. Another explanation might be that the modified histidine residue is not able to coordinate copper-B in the proper position relative to copper-A for catalysis to occur. If sulfite would add to Nε2, Nε2 would lose its ability to coordinate copper, because its lone pair of electrons would be engaged in the aromatic system of the imidazole ring. On the basis of this argument, and on the relative ease with which the sulfite moiety is lost upon MS fragmentation, we speculate that sulfite addition occurs on Nε2.
Mushroom tyrosinase, l-tyrosine, l-Dopa, NaHSO3, dithiothreitol, tropolone (2-hydroxy-2,4,6-cycloheptatrien-1-one), CuCl2 and EDTA were from Sigma Aldrich (St Louis, MO, USA). Kojic acid [5-hydroxy-2-(hydroxymethyl-4H-pyran-4-one] was from Acros Organics (Geel, Belgium), sequencing-grade pepsin was from Promega (Madison, WI, USA), sequencing-grade chymotrypsin was from Roche (Basel, Switzerland), and Glu1-fibrinopeptide was from Waters (Milford, MA, USA). UHPLC-MS-grade acetonitrile (ACN) and water were from Biosolve BV (Valkenswaard, The Netherlands). Water was prepared with a Milli-Q water purification system (Millipore, Billerica, MA, USA).
Purification of crude commercial mushroom tyrosinase
Crude commercial mushroom tyrosinase was purified by a single gel filtration step . A HiLoad 26/60 Superdex 200 column connected to an Akta Explorer system (GE Healthcare, Uppsala, Sweden) was used. Fifty milligrams of the enzyme, dissolved in 50 mm Hepes buffer (pH 6.8), was loaded and eluted with the same buffer at 4 mL/min. Fractions (5 mL) were collected, and activity was determined with a spectrophotometric assay: 50 μL of each fraction was combined with 100 μL of 0.8 mm tyrosine in a 96-well plate, and the absorbance at 520 nm was monitored over time. Active fractions were pooled and stored at −20 °C until further use. The purified mushroom tyrosinase had a specific activity of 23.3 ± 0.6 U/mg, where one unit (U) is defined as the amount of enzyme that catalyzes the formation of 1 μmol of dopachrome per minute at 25 °C (pH 6.5). Dopachrome formation was monitored spectrophotometrically at 475 nm with an extinction coefficient of 4770 m−1·cm−1 .
Samples were analyzed on an Accela UHPLC system (Thermo Scientific, San Jose, CA, USA) equipped with a pump, autosampler, and photodiode array detector.
Enzyme reaction products
Samples (5 μL) were injected onto a Hypersil Gold aQ column (2.1 × 150 mm; particle size 1.9 μm; Thermo Scientific). Water acidified with 0.1% (v/v) acetic acid (eluent A) and ACN acidified with 0.1% (v/v) acetic acid (eluent B) were used as eluents. The flow rate was 400 μL/min, and the temperature was controlled at 5 °C. The photodiode array detector was set to measure the range 200–600 nm. The following elution profile was used: 0–5 min, isocratic on 0% (v/v) B; 5–10 min, linear gradient from 0% to 35% (v/v) B; 10–10.1 min, linear gradient from 35% to 100% (v/v) B; 10.1–13 min, isocratic on 100% (v/v) B; 13–14 min, linear gradient from 100% to 0% (v/v) B; 14–19 min, isocratic on 0% (v/v) B.
Peptides from protease digests
Samples (5 μL) were injected onto an Acquity UPLC BEH Shield RP18 column (2.1 × 150 mm; particle size 1.7 μm; Waters). Water acidified with 0.1% (v/v) formic acid (eluent A) and ACN acidified with 0.1% (v/v) formic acid (eluent B) were used as eluents. The flow rate was 300 μL/min, and the temperature was controlled at 25 °C. The photodiode array detector was set to measure the range 200–600 nm. The following elution profile was used: 0–2 min, isocratic on 5% (v/v) B; 2–27 min, linear gradient from 5% to 50% (v/v) B; 27–27.1 min, linear gradient from 50% to 100% (v/v) B; 27.1–30 min, isocratic on 100% (v/v) B; 30–30.1 min, linear gradient from 100% to 5% (v/v) B; 30.1–35 min, isocratic on 5% (v/v) B.
Enzyme reaction products
MS data were obtained by analyzing samples on an LTQ-Velos (Thermo Scientific) equipped with a heated ESI probe coupled to the RP-UHPLC system. Nitrogen was used as sheath gas and auxiliary gas. Data were collected over the m/z range 150–500. Data-dependent MSn analysis was performed with a normalized collision energy of 35%. The MSn fragmentation was performed on the most intense product ion in the MSn − 1 spectrum. Most settings were optimized via automatic tuning with Tune Plus (xcalibur 2.1; Thermo Scientific). The system was tuned with l-Dopa in negative ionization mode. The source heater temperature was 100 °C, the transfer tube temperature was 300 °C, and the source voltage was 4 kV. Data acquisition and reprocessing were performed with xcalibur 2.1 (Thermo Scientific).
Peptides from protease digests
MS data were obtained by analyzing samples on an LTQ-VelosPro (Thermo Scientific) equipped with a heated ESI probe coupled to the RP-UHPLC system. Nitrogen was used as sheath gas and auxiliary gas. Data were collected over the m/z range 200–2000. Data-dependent MS2 analysis was performed with a normalized collision energy of 35%. The MS2 fragmentation was performed on the most intense ion in the preceding MS spectrum. A neutral loss of 40 in MS2 triggered subsequent MS3 fragmentation of the fragment ion showing the neutral loss of 40. Most settings were optimized via automatic tuning with Tune Plus (xcalibur2.1; Thermo Scientific). The system was tuned with the peptide Glu1-fibrinopeptide in positive ionization mode. The source heater temperature was 100 °C, the transfer tube temperature was 350 °C, and the source voltage was 4 kV. Data acquisition and reprocessing were performed with xcalibur 2.1 (Thermo Scientific). De novo sequencing of peptide fragmentation spectra was performed with peaksstudio 6.0 (Bioinformatics Solutions, Waterloo, Canada).
Oxygen consumption measurements
Oxygen consumption during the incubation of substrate with tyrosinase was measured with an Oxytherm System (Hansatech, Kings Lynn, UK). Incubations with l-Dopa (1 mm) or l-tyrosine (1 mm) and tyrosinase (0.075 U/mL), with or without NaHSO3 (1 mm), were performed in a total volume of 1 mL of 50 mm Hepes buffer (pH 6.5) at 25 °C. Data acquisition and analysis were performed with oxygraph plus software (Hansatech).
Pretreatment of tyrosinase with different inhibitors
Tyrosinase was preincubated with different inhibitors, as specified below. After incubation, inhibitors were removed by diafiltration with centrifugal filters [Amicon Ultra, 0.5 mL, 10-kDa molecular mass cut-off (Millipore)]. Filters were centrifuged (15 min, 14 000 g, 4 °C), after which the filtrate was discarded. The retentate (40 μL) was resuspended in 400 μL of 50 mm Hepes (pH 6.5), after which filters were centrifuged again. Six cycles of centrifugation and resuspension were performed. After the final centrifugation, the retentate was resuspended in the initial sample volume of 50 mm Hepes (pH 6.5).
Pretreatment of tyrosinase with NaHSO3/dithiothreitol/EDTA
Tyrosinase (6.4 U/mL) was incubated with NaHSO3 (1 mm) or dithiothreitol (1 mm) in 50 mm Hepes (pH 6.5) (1 h, 25 °C). Subsequently, NaHSO3 or dithiothreitol was removed by diafiltration, after which CuCl2 (1 mm) was added. After incubation (1 h, 25 °C), CuCl2 was removed by diafiltration.
Pretreatment of tyrosinase with NaHSO3 in the presence of competitive tyrosinase inhibitors
Tyrosinase (6.4 U/mL) was incubated with NaHSO3, tropolone, and kojic acid (each 1 or 10 mm), alone or in combination. After incubation (1 h, 25 °C), the inhibitors were removed by diafiltration.
Pretreatment of apo-tyrosinase with NaHSO3
Tyrosinase (6.4 U/mL) was incubated with dithiothreitol (10 mm) or EDTA (10 mm) in 50 mm Hepes (pH 6.5). After incubation (1 h, 25 °C), dithiothreitol or EDTA was removed by diafiltration, after which NaHSO3 (1 mm) was added. After incubation (1 h, 25 °C), NaHSO3 was removed by diafiltration, and CuCl2 (1 mm) was added. After incubation (1 h, 25 °C), CuCl2 was removed by diafiltration.
Protease digestion of NaHSO3-treated tyrosinase
Crude tyrosinase (2 mg/mL) was incubated with 100 mm NaHSO3 (1 h, 25 °C) in 50 mm Hepes (pH 6.5). After incubation, NaHSO3 was removed by diafiltration, as described above. After the last cycle of diafiltration, the samples were in 50 mm Hepes (pH 6.5), which was exchanged for the solution used for protease digestion [100 mm HCl (pH 1) if pepsin was used; 50 mm ammonium bicarbonate (pH 7.8) if chymotrypsin was used] by an additional two cycles of diafiltration. Pepsin or chymotrypsin was added in a ratio of 1 : 20 (w/w) to the NaHSO3-treated tyrosinase and to an untreated control. Samples were digested overnight at 37 °C.
We thank A. Westphal for technical support. This study was carried out with financial support from the Commission of the European Communities within the Seventh Framework Programme for Research and Technological Development (FP7), Grant Agreement 226930, title ‘Replacement of Sulphur Dioxide (SO2) in Food Keeping the Same Quality and Shelf-Life of the Products’, acronym SO2SAY.