Structural Characterization and In Vitro Antioxidant Activity of Metallothionein from Oratosquilla oratoria

We report here the purification of a novel metal-binding protein from Oratosquilla oratoria (O. oratoria MT-1) by gel and ion-exchange chromatography. SDS-PAGE and MALDI-TOF analyses demonstrated that isolated O. oratoria MT-1 was of high purity with a molecular weight of 12.4 kDa. The fluorescence response to SBD-F derivatives revealed that O. oratoria MT-1 contained a large number of sulfhydryl groups, which is a general property of metallothioneins. Zn and Cu metal stoichiometries for O. oratoria MT-1 were 3.97:1 and 0.55:1, respectively. The proportion of cysteine (Cys) residues in the amino acid composition was 32.69%, and aromatic amino acids were absent. The peptide sequence coverage with Macrobrachium rosenbergii calmodulin (accession AOA3S8FSK5) was 60%. Infrared spectroscopy of O. oratoria MT-1 revealed two obvious peaks at absorption frequencies for the amide I band and the amide II band. CD spectra revealed that the secondary structure was mainly composed of random coil (57.6%) and β-sheet (39.9%). An evaluation of in vitro antioxidant activity revealed that isolated O. oratoria MT-1 has strong reducing activities, exhibiting scavenging rates for DPPH and OH of 77.8% and 75.8%, respectively (IC50 values 0.57 mg/mL and 1.1 mg/mL). O. oratoria MT-1 may be used as a functional additive in cosmetics, health foods, and medical products, as well as a reference material for quantitative analysis of metallothionein in such products.


Introduction
Metallothioneins (MTs) are a family of low molecular weight (usually Mw < 10 kDa) Cys-rich (30-33% of total amino acids) proteins that are ubiquitous across many organisms [1][2][3][4][5][6][7]. MTs characteristically demonstrate strong resistance to thermal stress [8][9][10] and protease digestion [1,4]. The number and the location of Cys residues in natural MTs are conserved across many species, and these are organized as repeat arrangements of Cys-X-Cys or Cys-Cys motifs that bind Cu 2+ , Zn 2+ , or Cd 2+ [11]. Thus, MTs typically exhibit a strong metal binding capacity and a strong redox activity. MTs are primarily involved in the storage, transportation, and metabolism of trace metal elements; additional functions include detoxification and chelation of nonessential heavy metals, regulation of homeostasis, elimination of free radicals, protection of tissues from oxidative damage, and antagonism of ionizing radiation [12,13]. MTs also provide active centers for other metalloproteins and metalloenzymes [14]. MTs have been isolated from vertebrates, invertebrates, plants, and microorganisms, and the physicochemical properties of these isozymes are essentially the same [15]. Potentially useful MTs have also been isolated from several marine organisms, including sponges [16], mussels [17], shrimp [18], crabs [19], sea urchins [20], and fish [21].

Purification of O. oratoria MT-1
First, we obtained a crude fraction of the metal-binding proteins using gel filtration. The resulting gel filtration chromatogram on Sephadex G-50 is shown in Figure 1a. The response signals for Cu and Zn were significantly higher than those for Cd, Pb, and As (not shown in the figure due to their low responses). We observed a good correspondence between the ICP-MS signal responses (for Cu and Zn) and UV absorbance (at 270 nm and 220 nm) in the 16th-38th tubes. The collection tubes corresponding to this elution peak were pooled and then lyophilized. Since the UV absorption peak positions of MTs with bound metal are typically 270 m for Cu-MT, 250 nm for Cd-MT, and 220 nm for Zn-MT [35], we inferred that the metal-binding proteins prepared from O. oratoria were mainly in the form of Cu-MT and Zn-MT.
revealed that purified F1 exhibited the typical properties of an MT, in which a high Cys content, a large number of sulfhydryl groups [2,4,5], a strong metal binding capacity [3,11] and a strong redox activity [12,13] were all observed. The desalted F1 fraction was used as the high purity preparation of O. oratoria MT-1. Due to the limited yield (calculated by the obtained amount, the yield of fraction F2 was only 51.2% of F1), we have not prepared enough of fraction F2 for more follow-up structural characterization.

Molecular Weight Determination
SDS-PAGE analysis revealed that O. oratoria MT-1 and F2 migrated as single bands (Figure 2), indicating that the two prepared proteins were of high purity. Compared with the electrophoresis bands of marker Mw of O. oratoria MT-1 was calculated to be approximately 15 kDa, much higher than the MT expected value (typically Mw < 10 kDa) [4], while the Mw of F2 was about 8 kDa. Although the free sulfhydryl groups in Cys residues In general, the genes encoding MTs express tissue-specific subtypes due to polymorphisms [36]. Since Sephadex G-50 gel column chromatography could not separate the MT subtypes, DEAE Sepharose anion exchange chromatography was used to isolate subtypes from the crude O. oratoria metal-binding protein fraction. The elution curve for ion exchange chromatography is shown in Figure 1b. The 11th-20th tubes (peak F1) and the 33rd-40th tubes (peak F2) were separately pooled and subsequently lyophilized.
Next, Sephadex G-25 gel chromatography was used for further purification and desalination of F1 components (this was necessary, because a high-salt buffer was used to elute the sample in the previous step). The elution curve is shown in Figure 1c. The 15th-22nd fractions were combined and freeze-dried. Subsequent structural characterization revealed that purified F1 exhibited the typical properties of an MT, in which a high Cys content, a large number of sulfhydryl groups [2,4,5], a strong metal binding capacity [3,11] and a strong redox activity [12,13] were all observed. The desalted F1 fraction was used as the high purity preparation of O. oratoria MT-1. Due to the limited yield (calculated by the obtained amount, the yield of fraction F2 was only 51.2% of F1), we have not prepared enough of fraction F2 for more follow-up structural characterization.

Molecular Weight Determination
SDS-PAGE analysis revealed that O. oratoria MT-1 and F2 migrated as single bands (Figure 2), indicating that the two prepared proteins were of high purity. Compared with the electrophoresis bands of marker Mw of O. oratoria MT-1 was calculated to be approximately 15 kDa, much higher than the MT expected value (typically Mw < 10 kDa) [4], while the Mw of F2 was about 8 kDa. Although the free sulfhydryl groups in Cys residues of MTs are easily oxidized to form intermolecular disulfide bonds, resulting in molecular polymerization, this may not apply to the present study, because the loading buffer in SDS-PAGE contained a strong reducing agent, β-mercaptoethanol, which can break the disulfide bonds to obtain linear polypeptide chains [37]. However, the metal thiolate clusters in MTs may assist in making the protein structure in a compact form and limiting the loading of SDS on the surface of the protein, which can reduce the amount of negative charges in proteins, affecting their migration on electrophoresis and resulting in a higher apparent Mw [37][38][39][40]. Therefore, a more accurate method was required to calculate the precise Mw for O. oratoria MT-1. of MTs are easily oxidized to form intermolecular disulfide bonds, resulting in molecular polymerization, this may not apply to the present study, because the loading buffer in SDS-PAGE contained a strong reducing agent, β-mercaptoethanol, which can break the disulfide bonds to obtain linear polypeptide chains [37]. However, the metal thiolate clusters in MTs may assist in making the protein structure in a compact form and limiting the loading of SDS on the surface of the protein, which can reduce the amount of negative charges in proteins, affecting their migration on electrophoresis and resulting in a higher apparent Mw [37][38][39][40]. Therefore, a more accurate method was required to calculate the precise Mw for O. oratoria MT-1. MALDI-TOF spectrometric analysis of O. oratoria MT-1 in the mass-to-charge ratio (m/z) range of 500-30,000 is shown in Figure 3a, and Figure 3b demonstrates the regional enlarged view in m/z 8000-30,000. There were few miscellaneous peaks, indicating that the O. oratoria MT-1 preparation was highly pure; m/z 6190 was the double-charged molecular ion peak [M + 2H] 2+ for O. oratoria MT-1, while m/z 12,445 was the single-charge molecular ion peak [M + H] + . Based on the above results, the Mw of O. oratoria MT-1 was calculated as 12.4 kDa. A Zn-MT of similar Mw (14.4 kDa), used as a MT reference in the present work (named Rabbit liver MT-1, with the product batch of 002019120201), was obtained from rabbit liver by Dalian Free Trade Zone Lianhe Botai Biological Technology Co., Ltd. in China. Likewise, we previously reported that the molecular weights of two purified MTs (MT-M-I and MT-M-II) obtained from metal-exposed Arca subcrenata were both slightly > 17 kDa rather than the expected value (usually < 10 kDa) [32]. In addition, a purified Cu-MT isoform from Sinopotamon henanense was reported to exhibit an Mw of 13.9 kDa [34]. These diverse observations are consistent with the high Mw of O. oratoria MT-1 prepared here. These discrepancies may be explained by species specificity in MTs from marine organisms. Likewise, we previously reported that the molecular weights of two purified MTs (MT-M-I and MT-M-II) obtained from metal-exposed Arca subcrenata were both slightly >17 kDa rather than the expected value (usually <10 kDa) [32]. In addition, a purified Cu-MT isoform from Sinopotamon henanense was reported to exhibit an Mw of 13.9 kDa [34]. These diverse observations are consistent with the high Mw of O. oratoria MT-1 prepared here. These discrepancies may be explained by species specificity in MTs from marine organisms.

Fluorescence Derivatization of Sulfhydryl Groups
Since MTs have a much higher Cys content than most proteins, an assessment of the sulfhydryl groups would facilitate the development of a highly selective assay whose sensitivity would be enhanced by a reaction with a fluorogenic compound, 7-fluorobenzo-2oxa-1,3-diazole-4-sulfonic acid ammonium salt (SBD-F) [41,42]. An analysis by high-performance liquid chromatography (HPLC) coupled with a fluorescence detector (FLD) is shown in Figure 4. In the presence of O. oratoria MT-1 and the complete reaction solvent system (boric acid + ethylene diamine tetra acetic acid (EDTA) + Tris(2-carboxyethyl)phosphine (TCEP) + SBD-F), a high-intensity chromatogram peak was observed at approximately 4 min (Figure 4a). Chromatograms of the complete reaction solvent system (boric acid + EDTA +TCEP + SBD-F) itself and O. oratoria MT-1 with the reaction solvent system (boric acid + EDTA +TCEP) are shown in Figures 4b, 4c, respectively. Therefore, it can be inferred that a derivatization reaction happened between O. oratoria MT-1 and SBD-F and that a large number of sulfhydryl groups were constituent components of the prepared protein. These results are consistent with the general properties of MTs [2,4,5].

Fluorescence Derivatization of Sulfhydryl Groups
Since MTs have a much higher Cys content than most proteins, an assessment of the sulfhydryl groups would facilitate the development of a highly selective assay whose sensitivity would be enhanced by a reaction with a fluorogenic compound, 7-fluorobenzo-2-oxa-1,3-diazole-4-sulfonic acid ammonium salt (SBD-F) [41,42]. An analysis by highperformance liquid chromatography (HPLC) coupled with a fluorescence detector (FLD) is shown in Figure 4. In the presence of O. oratoria MT-1 and the complete reaction solvent system (boric acid + ethylene diamine tetra acetic acid (EDTA) + Tris(2-carboxyethyl)phosphine (TCEP) + SBD-F), a high-intensity chromatogram peak was observed at approximately 4 min (Figure 4a). Chromatograms of the complete reaction solvent system (boric acid + EDTA +TCEP + SBD-F) itself and O. oratoria MT-1 with the reaction solvent system (boric acid + EDTA +TCEP) are shown in Figure 4b,c, respectively. Therefore, it can be inferred that a derivatization reaction happened between O. oratoria MT-1 and SBD-F and that a large number of sulfhydryl groups were constituent components of the prepared protein.
These results are consistent with the general properties of MTs [2,4,5]. Molecules 2022, 27, x FOR PEER REVIEW 6 of 20

Analysis of Metal Stoichiometries
The metal content analyses for a commercially available MT (Rabbit liver MT-1) and purified O. oratoria MT-1 are detailed in Table 1. The Zn contents of Rabbit liver MTs and O. oratoria MT-1 were highest, followed by the Cu contents. Adjusting for the Mw of each

Analysis of Metal Stoichiometries
The metal content analyses for a commercially available MT (Rabbit liver MT-1) and purified O. oratoria MT-1 are detailed in Table 1. The Zn contents of Rabbit liver MTs and O. oratoria MT-1 were highest, followed by the Cu contents. Adjusting for the Mw of each protein, the Zn and Cu metal stoichiometries of Rabbit liver MT-1 were approximately 5.5:1 and 0.5:1. While the Zn stoichiometry of O. oratoria MT-1 was lower (3.97:1), the Cu stoichiometry (0.55:1) of O. oratoria MT-1 was comparable. These results mirrored the metal contents of O. oratoria muscle tissue (Zn > As > Cu; Table 2), indicating that purified O. oratoria MT-1 was mainly present in the Zn-MT and Cu-MT forms. In contrast, bound As metal was not detected in O. oratoria MT-1, probably reflecting the fact that As in marine organisms exists mainly as small organic-arsenic compounds, such as dimethylarsinic acid, arsenobetaine, arsenocholine, and arsenic sugar [43][44][45]. The Zn stoichiometry (approximately 4:1) of O. oratoria MT-1 was lower than that of MT-M-I and MT-M-II from Arca subcrenata (6:1 and 7:1) [33] and lower than that of an MT isoform from Homarus americanus, which exhibits a coordinating stoichiometry of six Zn 2+ or Cd 2+ [46]. The heterologous expressed MT of Sinopotamon henanense was observed to bind 15 equivalents of Zn 2+ in vivo (as analyzed by ESI-TOF-MS), but only six equivalents of Zn 2+ were required to form the stable metal-S coordination bonds in the tetrahedral structure that coordinates Zn [47]. In most cases, MTs bind 7-12 metal ions [48]. The relatively low Zn stoichiometry of O. oratoria MT-1 reported in the present study may be related to the use of ICP-MS for analysis, which has lower precision than ESI-TOF-MS. Conversely, Zn metal may be lost during isolation and purification because of unstable Zn 2+ binding to O. oratoria MT-1.

Amino Acid Composition
The amino acid compositions of O. oratoria MT-1 and Rabbit liver MT-1 are shown in Table 3. The Cys content was the highest in O. oratoria MT-1, accounting for 32.69% of the total amino acids. The observed Cys content of O. oratoria MT-1 was slightly higher than that of Rabbit liver MT-1 (31.99%). Aromatic amino acids (Phe, Trp, and Tyr) were not detected. This is a common feature of proteins in the MT family [4,5], contributing to our confidence in identifying O. oratoria MT-1 as an MT. were expressed by the grams of each amino acid per 100 g of O. oratoria MT-1. b Content ratio of each amino acid to the total amino acids. c "ND" indicated not detected (value < limit of detection). Data are presented as the means ± SD (n = 3).

Peptide Sequence Analysis
Successful identification of a peptide sequence by high-resolution mass spectrometry is dependent on the information integrity of proteins in the database. Therefore, choosing the appropriate species as a search condition is a key factor. At present, the protein sequence data for O. oratoria MT is not available. To expand the scope of our database comparison, crustaceans were selected as the relevant source for the comparison of O. oratoria MT-1 peptide sequences. In total, 560 peptides were identified in O. oratoria MT-1 after trypsin digestion. One of the highest matched proteins is shown in Table 4, and the details of the matching peptides are shown in Figure 5 and Table 5. The peptide sequence coverage was 60% for Macrobrachium rosenbergii calmodulin (accession AOA3S8FSK5). Calmodulins have a highly conserved molecular structure encoded in their protein sequences, and their biological activities are manifold, in addition to participating in various intracellular signal transduction pathways [49]. Since purified O. oratoria MT-1 exhibited high homology with calmodulin, it can be inferred that O. oratoria MT-1 may also possess calmodulin-like biological activities.      Through an online search of MTs on the UniProt website, it was found that the sequence information for MTs was mainly obtained from mammals, and no crustacean MT sequences were found. Therefore, the identification results for peptide sequence alignment of the prepared O. oratoria MT-1 are greatly affected by the limited database (which contains few crustacean proteins). While O. oratoria MT-1 and Macrobrachium rosenbergii calmodulin shared a high peptide sequence coverage, segments containing Cys residues were missing from the matched peptides. Thus, there is an apparent contradiction with the results of the amino acid composition analysis. However, the principles of the two detection methods are completely different. If the database had included the relevant protein information, the number of matched peptide sequences would be richer and more reliable. Nonetheless, Cys-rich peptides, including AWGSPCEICNPAECDCPIGFAK, CCYDTCLR, and ACNCLLLK, could be matched with other proteins. The matching of the peptide sequences to other proteins from the same or similar species should help us identify possible homologous proteins to O. oratoria MT-1, and it should provide information concerning potential biological functions.

Spectroscopic Characteristics of O. oratoria MT-1 2.7.1. IR(Infrared) Spectroscopy
As a rule, protein conformation is determined by regular secondary structures (α-helix, β-sheet, and β-turn) and irregular structures (loops and random coil) [50]. To analyze the O. oratoria MT-1 secondary structure, we performed IR spectroscopy. IR spectroscopy is a powerful tool that provides information on protein conformation and structural dynamics [51]. The infrared spectra of three standard amide bands are widely accepted as the diagnostic for specific secondary structure elements of a protein. These standard amide bands are the amide I band around 1700-1600 cm −1 (mainly C=O stretch), the amide II band around 1600-1500 cm −1 (C=N stretch and N-H bending vibration), and the amide III band around 1330-1220 cm −1 (N-H bending and C-N stretching vibration) [52]. In general, absorption band frequencies at 1600-1640 cm −1 are diagnostic of β-sheets, absorption band frequencies in the 1640-1650 cm −1 range are diagnostic of random coils, absorption band frequencies around 1650-1658 cm −1 are diagnostic of α-helices, and absorption band frequencies in the 1660-1695 cm −1 range are diagnostics of β-turns [53]. As demonstrated in Figure 6, the mid-infrared spectra (400-4000 cm −1 ) of O. oratoria MT-1 exhibited great similarities with Rabbit liver MT-1 (both in absorption frequencies and intensities). Two relatively strong bands at absorption frequencies of 1641 and 1540 cm −1 were observed in O. oratoria MT-1, representing amide I and amide II, respectively. The peak intensity of the amide III band (1245 cm −1 ) was weak, and a conspicuous band was found at 1074 cm −1 (stretching vibration of C-C and C-OH). The amide band I and amide band II of Rabbit liver MT-1 were found at 1644 and 1528 cm −1 , respectively, and the amide band III was also weak. Furthermore, the infrared spectra of both proteins exhibited a broad soft band around 3270 cm −1 (O-H stretching vibration). Together, the IR data provide further confirmation that O. oratoria MT-1 is a MT. Furthermore, the characteristic absorption peaks at 1640 cm −1 indicate that both O. oratoria MT-1 and Rabbit liver MT-1 contain a high percentage of a random coil secondary structure.
band around 1600-1500 cm −1 (C=N stretch and N-H bending vibration), and the amide III band around 1330-1220 cm −1 (N-H bending and C-N stretching vibration) [52]. In general, absorption band frequencies at 1600-1640 cm −1 are diagnostic of β-sheets, absorption band frequencies in the 1640-1650 cm −1 range are diagnostic of random coils, absorption band frequencies around 1650-1658 cm −1 are diagnostic of α-helices, and absorption band frequencies in the 1660-1695 cm −1 range are diagnostics of β-turns [53]. As demonstrated in Figure 6, the mid-infrared spectra (400-4000 cm −1 ) of O. oratoria MT-1 exhibited great similarities with Rabbit liver MT-1 (both in absorption frequencies and intensities). Two relatively strong bands at absorption frequencies of 1641 and 1540 cm −1 were observed in O. oratoria MT-1, representing amide I and amide II, respectively. The peak intensity of the amide III band (1245 cm −1 ) was weak, and a conspicuous band was found at 1074 cm −1 (stretching vibration of C-C and C-OH). The amide band I and amide band II of Rabbit liver MT-1 were found at 1644 and 1528 cm −1 , respectively, and the amide band III was also weak. Furthermore, the infrared spectra of both proteins exhibited a broad soft band around 3270 cm −1 (O-H stretching vibration). Together, the IR data provide further confirmation that O. oratoria MT-1 is a MT. Furthermore, the characteristic absorption peaks at 1640 cm −1 indicate that both O. oratoria MT-1 and Rabbit liver MT-1 contain a high percentage of a random coil secondary structure.

CD (Circular Dichroism) Spectroscopy
Protein CD spectra provide secondary structure information content by reporting on peptide bond absorption in the far ultraviolet region (185-245 nm). Specifically, an α-helical protein exhibits a positive peak near 192 nm and two negative shoulder peaks at 207-210 nm and 221 to 222 nm. β-sheet-containing proteins exhibit a negative peak at 216-218 nm and a positive peak at 185-200 nm. β-turn-containing proteins exhibit a positive peak around 205 nm, a weak negative peak at 220-230 nm, and a strong negative peak at 180-190 nm. Finally, the presence of a negative peak near 198 nm and a positive peak near 220 nm is characteristic of a random coil structure [54,55]. As shown in Figure 7, the CD spectrum of O. oratoria MT-1 exhibited a negative peak at 197.5 nm and a broad positive peak around 220 nm, which are typical characteristics of a random coil structure in proteins. In addition, a strong positive absorption peak at 191 nm and a small reversal peak at 216 nm provide evidence of the β-sheet secondary structure in O. oratoria MT-1. Characteristic absorption peaks for β-turn and α-helix were not present. Likewise, random coil and β-sheet were the predominant secondary structures in Rabbit liver MT-1. The proportions of each secondary structure in O. oratoria MT-1, as determined by the CD estimation program (Table 6), were 57.6% random coil, 39.9% β-sheet, 2.0% β-turn, and 0.6% α-helix. For comparison, the proportions for Rabbit liver MT-1 were 58.2% random coil, 28.0% β-sheet, 7.3% β-turn, and 6.5% α-helix ( Table 6). The total contents of α-helix, β-sheet, and β-turn in O. oratoria MT-1 and Rabbit liver in MT-1 were 42.4% and 41.8% (of the total secondary structure), respectively. These results suggest that the two proteins possess a relatively stable conformation. The proportions of the four secondary structure fractions (α-helix, β-sheet, β-turn, and random coil) dissolved in distilled water were determined using the protein secondary structure estimation program CDpro.

In Vitro Antioxidant Activity
In the presence of antioxidants, the purple color of a DPPH (1,1-diphenyl-2-picrylhydrazyl) solution fades, and this color change can be followed at 517 nm (the maximum absorption peak) [56]. Thus, the antioxidant capacity of a compound can be determined using DPPH absorption. As shown in Figure 8a, the DPPH scavenging rate of Vc (Vitamin C) was stable at 85.8-87.5% over the 0.4-1.5-mg/mL concentration range, with no significant difference between the concentrations (p > 0.05). The DPPH scavenging rates of O. oratoria MT-1 and Rabbit liver MT-1 increased significantly with a concentration in the 0.1-1.0-mg/mL range. At greater concentrations (1.0-1.5 mg/mL), the DPPH scavenging capacity of both O. oratoria MT-1 and Rabbit liver MT-1 were stable, with no significant difference between the concentrations (p > 0.05). The highest observed DPPH scavenging rates for O. oratoria MT-1 and Rabbit liver MT-1 were 77.8% and 73.1%, respectively. At identical concentrations, the DPPH scavenging rate of O. oratoria MT-1 was slightly higher than that of rabbit liver MT-1 (p > 0.05) but less than that of Vc (p < 0.05). The calculations indicate that the IC 50 of O. oratoria MT-1 activity against DPPH was 0.57 mg/mL, demonstrating that O. oratoria MT-1 had good antioxidant capacity.

In Vitro Antioxidant Activity
In the presence of antioxidants, the purple color of a DPPH (1,1-diphenyl-2-picrylhydrazyl) solution fades, and this color change can be followed at 517 nm (the maximum absorption peak) [56]. Thus, the antioxidant capacity of a compound can be determined using DPPH absorption. As shown in Figure 8a, the DPPH scavenging rate of Vc (Vitamin C) was stable at 85.8-87.5% over the 0.4-1.5-mg/mL concentration range, with no significant difference between the concentrations (p > 0.05). The DPPH scavenging rates of O. oratoria MT-1 and Rabbit liver MT-1 increased significantly with a concentration in the 0.1-1.0-mg/mL range. At greater concentrations (1.0-1.5 mg/mL), the DPPH scavenging capacity of both O. oratoria MT-1 and Rabbit liver MT-1 were stable, with no significant difference between the concentrations (p > 0.05). The highest observed DPPH scavenging rates for O. oratoria MT-1 and Rabbit liver MT-1 were 77.8% and 73.1%, respectively. At identical concentrations, the DPPH scavenging rate of O. oratoria MT-1 was slightly higher than that of rabbit liver MT-1 (p > 0.05) but less than that of Vc (p < 0.05). The calculations indicate that the IC50 of O. oratoria MT-1 activity against DPPH was 0.57 mg/mL, demonstrating that O. oratoria MT-1 had good antioxidant capacity. The OH (hydroxyl) radical is toxic to the human body, where it oxidizes both the protein and DNA and causes damage and necrosis to human cells [57]. As shown in Figure 8b, the hydroxyl radical scavenging rates of O. oratoria MT-1 and Rabbit liver MT-1 increased significantly with the protein concentrations in the 0.2-1.8-mg/mL concentration range (p < 0.05). In contrast, the hydroxyl radical scavenging rates were relatively stable in the 1.8-2.5-mg/mL concentration range (p < 0.05); the highest observed scavenging rates for O. oratoria MT-1 and Rabbit liver MT-1 were 75.8% and 72.1%, respectively. The hydroxyl radical scavenging rate of V C also demonstrated a significant dose-dependent relationship over the 0.2-1.2-mg/mL concentration range (p < 0.05). At higher V C concentrations, the scavenging rate stabilized; the highest scavenging rate was 90.2%. The IC 50 values of V C , O. oratoria MT-1, and Rabbit liver MT-1 were 0.38, 1.1, and 1.2 mg/mL, respectively. While the hydroxyl radical scavenging ability of O. oratoria MT-1 was lower than that of Vc, O. oratoria MT-1 exhibited good hydroxyl radical scavenging antioxidant ability.

Raw Materials
O. oratoria was purchased from the Zhoushan International Aquatic Market. All of the O. oratoria were caught in the sea area of the Zhoushan fishing ground (29°30′-31°00′ N, 120°30′-125°00′ E). The average body length was 15.5 (±0.5) cm, and the average body weight was 26.2 (±0.4) g. Before processing, surface impurities were washed away with deionized water. During processing, the shell was removed, and the edible parts were harvested and homogenized in a stainless-steel homogenizer (IKA Ultra-Turrax T-25, Staufen, Germany). The homogenates were then stored at −18 °C for future use.

Buffer Extraction
First, 20-mM Tris-HCl buffer solution (pH 8.0, containing 0.5-mM PMSF and 0.01% β-mercaptoethanol) was added to the sample in a 1:4 ratio. The suspension was stirred at 50 °C for 1 h and then centrifuged at 13,000 r/min for 25 min at 4 °C . The resulting supernatant was heated in a water bath at 80 °C for 3 min (Hitachi CR21G II centrifuge, Tokyo, Japan). The solution was subsequently cooled to room temperature and then centrifuged at 13,000 r/min for 25 min at 4 °C to collect the supernatant. After adding three times the volume of 4 °C precooled ethanol, the total protein was allowed to precipitate at −20 °C overnight. The resulting suspension was then centrifuged at 13,000 r/min for 25 min at 4 °C . Finally, the precipitate was dissolved in 3 mL of Tris-HCl buffer and lyophilized (SJIA- The reducing power of MTs can also be measured by analyzing the protein-dependent Fe 3+ -Fe 2+ transition. The potential antioxidant activity of a compound is known to mirror its reducing capacity, which has been demonstrated to exert antioxidant action by breaking the free radical chain [58,59]. As shown in Figure 8c, the reducing power of O. oratoria MT-1 was slightly higher than that of Rabbit liver MT-1, although both activities were weaker than that of V C . The reducing power of O. oratoria MT-1 demonstrated a good correlation with the protein concentration in the 0.2-1.8-mg/mL range but was observed to stabilize at higher concentrations. In our previous work, MT-M-I and MT-M-II prepared from Arca subcrenata also exhibited an increase in the DPPH and OH scavenging rates with the protein concentration. The DPPH scavenging rates for MT-M-I and MT-M-II were 85% and 72% (respectively) at a concentration of 100 µg/mL, and the OH scavenging rates were 57% and 60% (respectively) at a concentration of 50 µg/mL [32]. The DPPH scavenging rate for F1-2 prepared from Mizuhopecten yessoensis reached nearly 100% at a concentration of 100 µg/mL, while the OH scavenging rate was about 50% at a concentration of 50 µg/mL [33]. The in vitro antioxidant activities of these three MTs (from two different marine shellfish) were higher than that of O. oratoria MT-1. This apparent discrepancy can be explained by differences in the materials used and in the purification processes that can cause differences in the molecular structures of the prepared MTs (e.g., copurified inactivated protein). Moreover, the antioxidant activity assay used was different. Nonetheless, the in vitro antioxidant activity study demonstrated that the prepared O. oratoria MT-1 had good antioxidant capacity. 0.1-M Coomassie Brilliant Blue R-250 solution, and then scanned using a GelDoc XR gel imager (Bio-Rad, Hercules, CA, USA).

MALDI-TOF
O. oratoria MT-1 solution (1.0 mg/mL) was mixed with the matrix (15 g/L 3,5dimethoxy-4-hydroxycinnamic acid) at a volume ratio of 2:1 and then incubated at 37 • C for 6 h. Next, 2 µL of the mixed solution was spotted on the ground target plate of a MALDI-TOF MS system equipped with a 355 nm N 2 laser (Shimadzu MALDI-8020, Kyoto, Japan). All mass spectra were acquired in the 1.

Determination of Bound Metal Element
To determine the bound metal element, 1.0 mg/mL of O. oratoria MT-1 aqueous solution was directly injected into an ICP-MS instrument (Agilent 7900 ICP-MS, Santa Clara, CA, USA) for the quantitative analysis of Zn, Cu, Cd, Pb, and As.

Composition Analysis of Amino Acids
To analyze the amino acid composition, 20 mg of O. oratoria MT-1 was transferred to a 15-mL glass test tube, and 5 mL of 6 M HCl were added. The test tube was then sealed under nitrogen, and the sample was heated at 110 • C for 22 h to facilitate hydrolysis. After cooling to room temperature, the hydrolysate was transferred to a 10 mL volumetric flask, and the volume was adjusted to 10 mL with pure water. All samples were filtered through a 0.45 µm filter membrane, and the composition of 18 amino acids was analyzed using an automatic amino acid analyzer (Hitachi L-8900, Tokyo, Japan).

Peptide Sequence Analysis
To determine the peptide sequence, 200 µg of O. oratoria MT-1 was added to 200 µL of 8 M guanidine hydrochloride (dissolved in 50 mM phosphate solution, pH 8.0) and 5 µL of 1 M dithiothreitol (DTT). The resulting solution was thoroughly mixed and then incubated at 37 • C for 1 h. Next, 10 µL of 1 M iodoacetamide was added, and the sample was incubated at room temperature for 30 min in the dark. To facilitate solvent replacement with 50 mM phosphate solution (pH 8.0), the sample was centrifuged in a 15 mL ultrafiltration tube (molecular weight cut-off, 10 kDa) at 12,000 r/min for 30 min 4 • C. The filter was repeatedly rinsed with phosphate solution to maximize the recovery, and the sample volume was increased to 200 µL. Next, 50 µL of sample solution was digested with 1 µL of 1 mg/mL trypsin (sequencing grade) for 12 h at 37 • C. Finally, 1 µL of 10% FA was added to the sample, and the sample was mixed. Sequence analysis was subsequently performed on an EASY-nLC 1200 (Thermo, Waltham, MA, USA) coupled with a nano ESI Q-Exactive mass spectrometer (Thermo, Waltham, MA, USA). The MS/MS data obtained for O. oratoria MT-1 were analyzed and aligned in the NCBI blast database.

IR Spectroscopy
For IR analysis, 1.0 mg of lyophilized O. oratoria MT-1 was mixed with 100 mg of potassium bromide powder and pressed into tablets. The sample was then analyzed using a NICOLET-380 infrared spectrometer (Thermo, Waltham, MA, USA) under the following conditions: scanning spectral range, 400-4000 cm −1 ; resolution, 4 cm −1 ; repeated scanning times, 32; and subtraction background, air.

CD Spectroscopy
For CD analysis, O. oratoria MT-1 was first dissolved in distilled water (0.5 mg/mL). The samples were then analyzed at 25 • C using a JASCO-815 spectropolarimeter (JASCO, Tokyo, Japan) under the following conditions: scan speed, 100 nm/min; scan range, 190-260 nm; bandwidth, 0.5 nm; and length of quartz cell, 1.0 cm. CD spectra were corrected for solvent contributions and expressed in terms of specific ellipticities versus wavelength. The proportions of the secondary structure fractions (α-helix, β-sheet, βturn, and random coil) were determined using the protein secondary structure estimation program CDpro (Jasco, Tokyo, Japan).

DPPH Radical Scavenging Capacity
The DPPH radical scavenging capacity of O. oratoria MT-1 was measured according to the method of Huang et al. [60] with a slight modification. A volume of 500 µL of each protein sample at different concentrations was added to 500 µL of 0.1 mM DPPH (in ethanol). The mixtures were shaken and then incubated at 30 • C for 30 min in the dark. The absorbance at 517 nm (A s ) was then determined using a UV-3200 spectrophotometer (Mapada, Shanghai Meipuda instrument Co., Ltd., China). As a blank control (A 0 ), the absorbance of pure water was measured by the same method. Vc (Vitamin C) was used as the reference antioxidant. The DPPH radical scavenging capacity was calculated using the formula: DPPH radical scavenging capacity (%) = (1 − A s /A 0 ) × 100.

OH Radical Scavenging Capacity
The hydroxyl radical scavenging capacity of O. oratoria MT-1 was determined as described by Han et al. [61] with a slight modification. First, 2 mL of protein sample (at different concentrations), 2 mL of 6 mM ferrous sulfate solution, and 2 mL of 6 mM salicylic acid (in ethanol) were added in turn to a 10 mL test tube. After thorough mixing, 2 mL of 6 mM H 2 O 2 solution was added to initiate the reaction, and the sample was incubated at 37 • C for 30 min in the dark. The absorbance was then evaluated at 510 nm. Vc was used as the reference antioxidant. The hydroxyl radical scavenging capacity was calculated according to the following formula: OH scavenging capacity (%) = [1 − (As − As 0 )/A 0 ] × 100, where As represents the absorbance of the protein sample, As 0 represents the absorbance of the blank control group, and A 0 represents the absorbance of the protein sample without hydrogen peroxide.

Reducing Power
The reducing power of O. oratoria MT-1 was determined as described by Gu et al. [62] with a slight modification. First, 2 mL of protein sample (at different concentrations), 2 mL of 0.2 M PBS (pH 6.6), and 2 mL of 1% potassium ferricyanide solution were added in turn to a test tube. After thorough mixing, the samples were incubated at 50 • C for 30 min. Upon cooling to room temperature, 2 mL of 10% trichloroacetic acid were added. The mixture was then centrifuged at 3000 r/min for 10 min. A 3 mL aliquot of the supernatant was added to 3 mL of pure water and 0.5 mL of 0.1% ferric chloride. After shaking, the mixture was allowed to stand at room temperature for 10 min. Finally, the absorbance was determined at 700 nm. The reducing ability was expressed by the absorbance value, and Vc was used as the reference antioxidant.

Statistical Analysis of Data
Analyses were performed in triplicate, and the results were expressed as the mean ± standard deviation. SPSS 16.0 software (IBM, Armonk, NY, USA) was used for the statistical analyses of the test results. The Duncan multiple comparison method was used for the significance analysis. A p < 0.05 indicates a significant difference in the results. Excel 2010 (Microsoft, Redmond, WA, USA) and Origin8.0 software (OriginLab, Northampton, MA, USA) were used to draw and process the charts.

Conclusions
In the present study, we reported the purification and characterization of O. oratoria MT-1, which is a novel metal-binding protein. Structure characterization revealed that O. oratoria MT-1 displayed the typical structural features of a metallothionein. Thus, O. oratoria MT-1 was present in the Zn-MT and Cu-MT forms, its Cys content was around 32.69%, and the proportions of each secondary structure were random coil (57.6%) > β-sheet (39.9%) > β-turn (2.0%) > α-helix (0.6%). An evaluation of its in vitro antioxidant capacity revealed that O. oratoria MT-1 demonstrated strong reducing power and good scavenging capacity for DPPH and hydroxyl radicals. Due to its biological activities in binding heavy metals and scavenging free radicals, O. oratoria MT-1 has good application potential as an additive in cosmetics and health food; it can also be used as a reference material for the quantitative analysis of MT in such products.
However, there were also some imperfections in the present work that need to be considered in the future, such as the facts that O. oratoria MT-1 was still not fully characterized and that the structural information about the F2 fraction was still unknown. Future studies must consider the accurate protein sequence, tertiary structures, and the binding sites of metal elements in protein molecules. Moreover, animal models to study the antioxidant activities of O. oratoria MT-1 or F2 fraction in vivo must be established to provide basic data and theoretical support for their industrial application.