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Article

The Heme Cavity Is Essential for the Peroxidase and Antibacterial Activity of Homodimer Hemoglobin from the Blood Clam Tegillarca granosa

by
Lili Pu
1,
Shuting Dai
1,
Zongming Wu
1,
Sufang Wang
1,2,* and
Yongbo Bao
1,2,*
1
College of Biological and Environmental Sciences, Zhejiang Wanli University, Ningbo 315100, China
2
Ninghai Institute of Mariculture Breeding and Seed Industry, Zhejiang Wanli University, Ninghai 315604, China
*
Authors to whom correspondence should be addressed.
Fishes 2024, 9(12), 512; https://doi.org/10.3390/fishes9120512 (registering DOI)
Submission received: 4 November 2024 / Revised: 9 December 2024 / Accepted: 13 December 2024 / Published: 15 December 2024
(This article belongs to the Special Issue Shellfish Genetics and Breeding for Aquaculture)
Browse Figures
Figure 1
<p>Effects of SDS on the peroxidase and antibacterial activities of Tg-HbI. (<b>a</b>) Inactivation of Tg-HbI in the presence of SDS. (<b>b</b>) Effects of SDS on the antibacterial activity of Tg-HbI against <span class="html-italic">B. subtilis</span>. 1, SDS; 2, Tg-HbI; 3, mixed solution of Tg-HbI and SDS.</p> "> Figure 2
<p>Effect of SDS on the fluorescence of Tg-HbI. (<b>a</b>) Alterations in the intrinsic fluorescence emission spectra of Tg-HbI in the presence of SDS. (<b>b</b>) Intrinsic fluorescence intensity changes. (<b>c</b>) Maximum emission wavelength changes. (<b>d</b>) Alterations in the ANS binding fluorescence spectra of Tg-HbI in the presence of SDS. (<b>e</b>) ANS fluorescence intensity changes. (<b>f</b>) Alterations in the maximum emission wavelength of ANS.</p> "> Figure 3
<p>Effect of SDS on the UV-Vis absorbance spectra of Tg-HbI. (<b>a</b>) Alterations in the UV-Vis absorbance spectra of Tg-HbI in the presence of SDS. (<b>b</b>) Maximum absorbance changes. (<b>c</b>) Maximum absorption wavelength changes.</p> "> Figure 4
<p>Minimum energy docked pose of the complex with SDS and Tg-HbI. (<b>a</b>) Overall structure of SDS in complex with Tg-HbI. The structure of the Tg-HbI subunit displayed in cartoon form. SDS is colored green. (<b>b</b>) 3D interaction of SDS with the active site pocket of Tg-HbI. The key residues involved in ligand binding are shown as blue sticks. Yellow dashed lines represent H-bonds. (<b>c</b>) 2D diagram of intermolecular interactions. H-bonds are depicted as green dashed lines. Residues involved in hydrophobic interactions are shown as the spoked arcs.</p> ">
Versions Notes

Abstract

:
This study investigates the essential role of the heme cavity in the peroxidase and antibacterial activities of homodimeric hemoglobin (Tg-HbI) from the blood clam Tegillarca granosa. After treatment with sodium dodecyl sulfate (SDS), the peroxidase and antibacterial activities of the Tg-HbI were significantly inhibited, with the degree of inhibition correlating positively with the SDS concentration. Fluorescence spectroscopy, UV-Vis spectroscopy, and molecular docking analysis further revealed that SDS interacts with key amino acid residues (e.g., His70 and His102) in the heme cavity of Tg-HbI, causing conformational changes that disrupt the internal hydrophobic interactions, thus inhibiting its function. This study confirms that the antibacterial effect of Tg-HbI is mediated through its peroxidase activity and that the heme cavity plays a critical role in maintaining this activity. These findings lay a foundation for further research on the immune defense functions of hemoglobin and provide new insights into the mechanisms of environmental adaptation in T. granosa.
Keywords:
Tegillarca granosa hemoglobin; heme cavity; peroxidase activity; antibacterial activity; sodium dodecyl sulfate
Key Contribution: This study reveals that the heme-binding cavity is the structural basis for the Tg-HbI to maintain its peroxidase and antibacterial activity by multi-spectroscopic techniques and molecular docking.

1. Introduction

Tegillarca granosa, commonly known as the blood clam, belongs to the phylum Mollusca, class Bivalvia, and family Arcidae. Unlike most bivalves such as Solen strictus and Ruditapes philippinarum, whose blood is colorless, blood clams possess an abundance of red blood, hence their name [1]. This red coloration in T. granosa is due to the presence of hemoglobin (Hb), which exists in two forms: a homodimeric Tg-HbI and a heterotetrameric Tg-HbII [2,3,4]. In vertebrates, the dimeric HbI has long been replaced by the more efficient oxygen-carrying tetrameric HbII [5,6]. However, in blood clams, even though the Tg-HbII has evolved, Tg-HbI has been retained and constitutes 40% of the total Hb [7]. This raises intriguing questions about the function of Tg-HbI. Early studies have shown that Tg-HbI has a stronger oxygen-binding capacity and antibacterial activity than Tg-HbII [8], suggesting that Tg-HbI plays a critical role in the hypoxia tolerance and immune defense of blood clams, helping them adapt to harsh environments.
In 1958, Hobson first discovered the antibacterial properties of bovine Hb [9]. Subsequent studies revealed that Hb from multiple species, including humans, crocodiles, horses, snakes, and blood clams, exhibited broad-spectrum antibacterial activity against Gram-negative bacteria, Gram-positive bacteria, and fungi [10,11,12,13,14,15]. Despite these findings, the antibacterial role of Hb, especially its mechanisms, has received limited attention due to the well-developed immune systems of vertebrates.
Recent studies have demonstrated that Hb exhibits “pseudoperoxidase” or peroxidase-like activity in bacteria [16], invertebrates [4,17], and humans [18,19,20,21]. It has been suggested that human Hb may exert antibacterial effects through its peroxidase activity, which catalyzes the production of reactive oxygen species (ROS) [12,19]. Moreover, microbial extracellular proteases and pathogen-associated molecular patterns have been shown to enhance Hb’s peroxidase activity, promoting the generation of superoxide anions (O2−) [21,22,23,24]. Our previous research has also demonstrated that the inhibition of peroxidase activity by Cu2+ reduces the antibacterial activity of Tg-HbI purified from blood clam hemocytes [25]. These findings suggest that the antibacterial function of Tg-HbI may be linked to its peroxidase activity.
Structurally, Tg-HbI’s heme cavity shares similarities with natural peroxidases, such as horseradish peroxidase (HRP) and myeloperoxidase (MPO), including the presence of b-type heme and conserved amino acids like proximal His, distal His, and Arg [4,17]. Our previous studies hypothesized that proline might protect the iron porphyrin and the heme cavity structure from Cu2+-induced damage [25], thereby preventing Tg-HbI inactivation. Therefore, we propose that the heme cavity of Tg-HbI plays a crucial role in maintaining its antibacterial and peroxidase activity.
To test this hypothesis, we used sodium dodecyl sulfate (SDS), an anionic surfactant, as a model. SDS interacts with proteins through hydrophobic interactions [26,27,28,29,30], allowing us to investigate the relationship between the Tg-HbI’s heme cavity, peroxidase activity, and antibacterial properties. By examining the effects of SDS on Tg-HbI, we aim to determine whether Hb’s peroxidase activity is involved in its antibacterial function and to explore the structural basis of Tg-HbI’s catalytic activities, laying the groundwork for further studies on Tg-HbI’s role in the immune defense of blood clams.

2. Materials and Methods

2.1. Peroxidase Activity Assay

The Tg-HbI was purified from T. granosa following our previously reported method [8], and the peroxidase activity was measured as described previously [31]. Different concentrations of SDS (Solarbio, Beijing, China) were mixed with an equal volume of Tg-HbI, and the final SDS concentrations were 0.25, 0.5, 0.75, 1.0, 1.5, 2.0, 2.5, 3.0, 4.0, 5.0, 6.0, 7.0, and 8.0 mM, respectively. After incubation at 25 °C for 2 h, a 10 µL sample of the reaction mixture was added to 1 mL of the substrate solution containing 4 mM guaiacol (Macklin, Shanghai, China), 2 mM hydrogen peroxide (Aladdin, Shanghai, China), and 50 mM phosphate-buffered saline (PBS) buffer. The cuvette was covered with seal film and shaken by hand several times to mix the two liquids thoroughly. The absorbance was then recorded at 470 nm using a UV-1800 spectrophotometer (Shimadzu, Kyoto, Japan). Each assay was repeated in triplicate.

2.2. Antibacterial Activity Assay

The effects of SDS on the antibacterial activity of the Tg-HbI were evaluated using the agar diffusion method. Approximately 15 mL of molten LB medium was poured into a sterile Petri dish. After the medium solidified, 100 μL of B. subtilis (Strain NO. ATCC 6633, Solarbio, Beijing, China) suspension (107 CFU/mL) was evenly spread on the surface of the agar plate. Three sterilized Oxford cups were placed equidistant in the Petri dish. The experimental group comprised Tg-HbI blended with SDS in equal volumes, and Tg-HbI and SDS served as the positive and negative control, respectively. The final concentration of SDS was 2 mM. After incubation at 37 °C for 2 h, the mixture of Tg-HbI and SDS, the positive control, and the negative control were dropped into the individual Oxford cups on the agar plate, respectively. The plate was incubated overnight at 37 °C. The antibacterial activity of the different samples was determined by observing the size of the inhibition zone. Petri dishes, Oxford cups, and other materials were all sterilized in an autoclave prior to use.

2.3. Intrinsic Fluorescence Spectroscopy Assay

The Tg-HbI and 50 mM PBS solution (pH 7.2) containing varying concentrations of SDS were mixed thoroughly by shaking. The mixtures were subsequently left in the dark at 25 °C for 2 h prior to measurements. The fluorescence spectra of the enzyme incubation mixtures were recorded from 300 to 400 nm with an excitation wavelength of 280 nm using an F-2500 spectrofluorometer (Hitachi, Tokyo, Japan). Excitation and emission slit widths were 10 nm, and a scan speed of 1200 nm/min was used. All fluorescence spectra were corrected based on the corresponding protein-free sample.

2.4. ANS Fluorescence Spectroscopy Assay

To explore the effect of SDS on the Tg-HbI hydrophobic region, we incubated Tg-HbI samples with various SDS concentrations for 1.5 h, then incubated the samples with 3 μL of 40 μM ANS (Sigma, Shanghai, China) for 30 min in the dark before recording the spectra using a Hitachi F-2500 spectrofluorometer. The excitation wavelength for 1-anilinonaphthalene-8-aulfonic acid (ANS) binding fluorescence was 390 nm, and the emission was observed between 420 and 600 nm. The scan speed was 1200 nm/min, the slits were adjusted to 10 nm, and the electric voltage was set at 500 V. All resultant spectra were collected at 25 °C.

2.5. UV-Vis Spectroscopy

The UV-Vis spectra of the Tg-HbI were recorded using a Shimadzu UV-1800 spectrophotometer. After incubation with various SDS concentrations for 2 h, the UV-Vis absorption spectra of Tg-HbI were scanned using a 1 cm quartz cell in the range of 250 to 650 nm at 25 °C. All measurements were repeated in triplicate, and the mean values were calculated.

2.6. Molecular Docking

The predicted molecular models of the Tg-HbI were built by the SWISS-MODEL database (http://swissmodel.expasy.org/) (accessed on 25 October 2024) using the X-ray crystal structure of HbI (81.6% sequence identity; PDB ID: 3g53) from Scapharca inaequivalvis as a template. The three-dimensional (3D) structure of SDS (PubChem CID: 3423265) was obtained from the PubChem Database (https://pubchem.ncbi.nlm.nih.gov/) (accessed on 26 October 2024). AutoDock Vina 1.1.2 [32] was used to simulate the interaction between the Tg-HbI and SDS and to predict the binding sites. The best possible docked mode was selected according to the lowest binding energy. PyMOL 3.0.5 software was applied to visualize the docking conformation. The interactions of the best-docked molecules were analyzed using LigPlot 2.2.4 [33].

3. Results

3.1. Effects of SDS on the Peroxidase and Antibacterial Activities of Tg-HbI

The relative peroxidase activity of the Tg-HbI decreased as the SDS concentration increased, and SDS exerted a significant inhibitory effect, even at low concentrations (Figure 1a). When the concentration of SDS was 2 mM, the enzyme activity of Tg-HbI was only 15% of the original, and the antibacterial activity of Tg-HbI against B. subtilis was lost in the agar diffusion assay (Figure 1b).

3.2. Effects of SDS on the Fluorescence Spectra of Tg-HbI

Intrinsic fluorescence is sensitive to changes in the polarity around the Trp residue, which provides information about the interior structural perturbations of proteins [34,35,36]. We found that the fluorescence intensity of the Tg-HbI was enhanced and that its maximum emission wavelength showed an obvious blue shift with increasing concentrations of SDS (Figure 2a,c). When using SDS concentrations up to 4 mM, an increase in the maximum fluorescence intensity to approximately 3209 a.u. was observed, which was significantly higher than the native state (a value of 1453 a.u.) (Figure 2b).
We expected that the SDS-induced unfolding of the Tg-HbI would expose buried non-polar amino acids that could be probed by ANS binding fluorescence [37,38]. We found that the ANS fluorescence intensity of Tg-HbI fluctuated at the same level as the native folded state at SDS concentrations ≤ 2 mM, but it was enhanced at concentrations ranging from 3 to 5 mM (Figure 2d,e). The maximum emission wavelength of Tg-HbI with increasing SDS concentration red-shifted from 475 to 491 nm (Figure 2f). These results suggested that an increasing number of ANS were bound to the hydrophobic residues of the Tg-HbI.

3.3. Effects of SDS on the UV-Vis Spectra of Tg-HbI

The UV-Vis spectra of the Tg-HbI were characterized by three absorption bands (Figure 3a). The weak absorption peak at 280 nm is due to aromatic residues (Trp, Tyr, and Phe) [39]. The Soret band near 414 nm is often used to characterize changes between the porphyrin ring and the conformation of a protein, whereas the pair of Q bands at 500–600 nm is related to the iron porphyrin [40,41]. As the SDS concentration increased, the absorption peak at 280 nm showed altered absorbance, which suggested a change in the microenvironment of the Tyr and Trp residues in the Tg-HbI. The intensity of the Soret band decreased with little change in peaks (Figure 3b,c), indicating that SDS affected the interaction between the heme and amino acid residues in the heme cavity. The changes in the shape of the Q band in the 2–3 mM SDS exposure groups were pronounced, and the characteristic absorption peak disappeared.

3.4. Molecular Docking of Tg-HbI and SDS

We conducted a computer simulation study to better understand the interaction of the Tg-HbI and SDS at the molecular level and to clearly characterize the effects of SDS on the structure of Tg-HbI. Figure 4 shows the lowest energy conformation with a binding energy of −6.0 kcal/mol for SDS binding to Tg-HbI. The alkyl tail of the SDS molecule was inserted into the central cavity of Tg-HbI and formed extensive hydrophobic interactions with nine amino acid residues (Figure 4a, Table 1). The proximal and distal His, the essential residues for a Tg-HbI with peroxidase activity, were also found to be involved in the interaction between the Tg-HbI and SDS (Figure 4b). The side chain of His70 and His102 formed two hydrogen (H)-bonds with the head group of the SDS (bond length: 3.14 Å and 3.01 Å, respectively), which enhanced the binding force (Figure 4c). These findings suggested that SDS occupied the heme cavity of Tg-HbI and disrupted the internal hydrophobic interactions, which supported the results of UV-Vis and fluorescence spectroscopy.

4. Discussion

Our experimental results demonstrate that SDS significantly inhibits both the peroxidase and antibacterial activity of Tg-HbI, and this inhibitory effect is dose-dependent on the concentration of SDS. At an SDS concentration of 2 mM, the Tg-HbI retained only 15% of its original peroxidase activity and completely lost its antibacterial activity against B. subtilis in the agar diffusion assay. This observation is consistent with previous studies on Hb, such as Cu2+ inhibition studies [25], which also showed a loss of antibacterial activity when peroxidase activity was suppressed. These findings support the hypothesis that Hb exerts its antibacterial effect by generating ROS through its peroxidase activity [11,12,23,42].
Previous studies have indicated that seven conserved residues in the heme-binding cavity of HbI from T. granosa and S. inaequivalvis, namely Met38, Arg54, His70, Lys97, Asn101, His102, and Arg105, are essential for heme binding [43]. Several residues located near the distal side of the heme in HRP, specifically His and Arg, are known to be crucial for the enzyme’s high peroxidase-like activity [44,45]. Regarding the distal His, having a suitable distance between its nitrogen atom and the heme iron could enhance the peroxidase activity of heme proteins [46]. The observed peroxidase activity in Tg-HbI may thus be attributed to the presence of a heme-binding pocket. Through fluorescence spectroscopy analysis and molecular docking studies, we further investigated how SDS affects the structure and function of Tg-HbI. As the concentration of SDS increased, the fluorescence intensity of the Tg-HbI was significantly enhanced, with a noticeable blue shift, indicating that SDS induced conformational changes in Tg-HbI, leading to the exposure of its hydrophobic regions [30,47,48]. This exposure likely disrupted the native hydrophobic interactions that are critical for maintaining the structural integrity of Tg-HbI [28,49]. Molecular docking analysis also showed that the alkyl tail of SDS inserted into the heme cavity of the Tg-HbI, forming hydrophobic interactions with several key amino acid residues and strong H-bonds with His70 and His102, the core residues for its peroxidase activity [50]. These results suggest that SDS occupies hydrophobic sites within the heme cavity, disrupting the iron-protein interactions that stabilize the heme group [51,52].
When the SDS concentration exceeded 2 mM, the Tg-HbI underwent significant conformational changes, including further fluorescence blue shifts and greater exposure of its heme cavity, reinforcing the destructive effects of SDS on its activity and ultimately leading to the loss of Tg-HbI’s antibacterial functions in the agar diffusion assay. Compared to the tetrameric Tg-HbII, the Tg-HbI has stronger peroxidase activity and antibacterial effects [7], which may be related to subtle differences in their heme cavity structures.
This study not only highlights the critical role of the heme cavity in maintaining the Tg-HbI’s peroxidase and antibacterial activities but also provides new insights into the immune defense function of Tg-HbI in bivalve molluscs T. granosa. Compared to previous studies that primarily focused on the oxygen transport function of Hb, our research places more emphasis on the structural basis of Tg-HbI’s antibacterial function. Specifically, we have confirmed the hypothesis that the Tg-HbI exerts its antibacterial effect through peroxidase activity, and we demonstrated the importance of the heme cavity in maintaining this function under stress conditions, such as SDS exposure. Future studies could employ three-dimensional structural analysis and molecular dynamics simulations to further investigate the specific molecular mechanisms underlying Tg-HbI’s adaptation to extreme environments, such as hypoxia or high salinity, which are characteristic of the habitats of T. granosa.

5. Conclusions

This study highlights the dependence of the Tg-HbI’s antibacterial function on its peroxidase activity and the structural integrity of its heme cavity, which is highly sensitive to environmental stressors like SDS. By revealing how structural disruption impairs Tg-HbI’s function, our findings deepen our understanding of its role in immune defense and provide a basis for exploring its adaptations to extreme environments. Future research employing advanced structural and computational approaches could further elucidate the molecular mechanisms underlying Tg-HbI’s versatility, offering valuable insights into Hb multifunctionality in marine organisms.

Author Contributions

Methodology and experiments, L.P., Z.W. and S.W.; software and visualization, L.P. and S.D.; data analyses, L.P., S.D. and Z.W.; writing—original draft preparation, L.P.; writing—review and editing, S.W. and Y.B.; project administration, S.W. and Y.B.; and funding acquisition, S.W. and Y.B. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Zhejiang Major Program of Science and Technology (2021C02069-7), the Ningbo Public Welfare Science and Technology Program (2023S086), the Key Natural Science Foundation of Ningbo (2023J042), and the Zhejiang Provincial Top Key Discipline of Biological Engineering (ZS2021008).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

We share our research data.

Conflicts of Interest

The authors declare no conflicts of interest. The funders had no role in any of the following: the design of this study; the collection, analyses, or interpretation of data; the writing of the manuscript; or in the decision to publish the results.

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Figure 1. Effects of SDS on the peroxidase and antibacterial activities of Tg-HbI. (a) Inactivation of Tg-HbI in the presence of SDS. (b) Effects of SDS on the antibacterial activity of Tg-HbI against B. subtilis. 1, SDS; 2, Tg-HbI; 3, mixed solution of Tg-HbI and SDS.
Figure 1. Effects of SDS on the peroxidase and antibacterial activities of Tg-HbI. (a) Inactivation of Tg-HbI in the presence of SDS. (b) Effects of SDS on the antibacterial activity of Tg-HbI against B. subtilis. 1, SDS; 2, Tg-HbI; 3, mixed solution of Tg-HbI and SDS.
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Figure 2. Effect of SDS on the fluorescence of Tg-HbI. (a) Alterations in the intrinsic fluorescence emission spectra of Tg-HbI in the presence of SDS. (b) Intrinsic fluorescence intensity changes. (c) Maximum emission wavelength changes. (d) Alterations in the ANS binding fluorescence spectra of Tg-HbI in the presence of SDS. (e) ANS fluorescence intensity changes. (f) Alterations in the maximum emission wavelength of ANS.
Figure 2. Effect of SDS on the fluorescence of Tg-HbI. (a) Alterations in the intrinsic fluorescence emission spectra of Tg-HbI in the presence of SDS. (b) Intrinsic fluorescence intensity changes. (c) Maximum emission wavelength changes. (d) Alterations in the ANS binding fluorescence spectra of Tg-HbI in the presence of SDS. (e) ANS fluorescence intensity changes. (f) Alterations in the maximum emission wavelength of ANS.
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Figure 3. Effect of SDS on the UV-Vis absorbance spectra of Tg-HbI. (a) Alterations in the UV-Vis absorbance spectra of Tg-HbI in the presence of SDS. (b) Maximum absorbance changes. (c) Maximum absorption wavelength changes.
Figure 3. Effect of SDS on the UV-Vis absorbance spectra of Tg-HbI. (a) Alterations in the UV-Vis absorbance spectra of Tg-HbI in the presence of SDS. (b) Maximum absorbance changes. (c) Maximum absorption wavelength changes.
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Figure 4. Minimum energy docked pose of the complex with SDS and Tg-HbI. (a) Overall structure of SDS in complex with Tg-HbI. The structure of the Tg-HbI subunit displayed in cartoon form. SDS is colored green. (b) 3D interaction of SDS with the active site pocket of Tg-HbI. The key residues involved in ligand binding are shown as blue sticks. Yellow dashed lines represent H-bonds. (c) 2D diagram of intermolecular interactions. H-bonds are depicted as green dashed lines. Residues involved in hydrophobic interactions are shown as the spoked arcs.
Figure 4. Minimum energy docked pose of the complex with SDS and Tg-HbI. (a) Overall structure of SDS in complex with Tg-HbI. The structure of the Tg-HbI subunit displayed in cartoon form. SDS is colored green. (b) 3D interaction of SDS with the active site pocket of Tg-HbI. The key residues involved in ligand binding are shown as blue sticks. Yellow dashed lines represent H-bonds. (c) 2D diagram of intermolecular interactions. H-bonds are depicted as green dashed lines. Residues involved in hydrophobic interactions are shown as the spoked arcs.
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Table 1. Binding parameters and amino acid residues involved in Tg-HbI–SDS interaction.
Table 1. Binding parameters and amino acid residues involved in Tg-HbI–SDS interaction.
Hydrophobic InteractionsDistance (Å)H-Bond InteractionsBinding Energy 1 (kcal/mol)Protein-Ligand Complex
Met38, Leu41, Thr48, Phe52, Leu74, Ile107, Glu111, Phe112, Ile1153.14 Å
3.01 Å		His70 N-H…O
His102 N-H…O		–6.0Tg-HbI-SDS
1 The binding energy was evaluated using AutoDock.
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MDPI and ACS Style

Pu, L.; Dai, S.; Wu, Z.; Wang, S.; Bao, Y. The Heme Cavity Is Essential for the Peroxidase and Antibacterial Activity of Homodimer Hemoglobin from the Blood Clam Tegillarca granosa. Fishes 2024, 9, 512. https://doi.org/10.3390/fishes9120512

AMA Style

Pu L, Dai S, Wu Z, Wang S, Bao Y. The Heme Cavity Is Essential for the Peroxidase and Antibacterial Activity of Homodimer Hemoglobin from the Blood Clam Tegillarca granosa. Fishes. 2024; 9(12):512. https://doi.org/10.3390/fishes9120512

Chicago/Turabian Style

Pu, Lili, Shuting Dai, Zongming Wu, Sufang Wang, and Yongbo Bao. 2024. "The Heme Cavity Is Essential for the Peroxidase and Antibacterial Activity of Homodimer Hemoglobin from the Blood Clam Tegillarca granosa" Fishes 9, no. 12: 512. https://doi.org/10.3390/fishes9120512

APA Style

Pu, L., Dai, S., Wu, Z., Wang, S., & Bao, Y. (2024). The Heme Cavity Is Essential for the Peroxidase and Antibacterial Activity of Homodimer Hemoglobin from the Blood Clam Tegillarca granosa. Fishes, 9(12), 512. https://doi.org/10.3390/fishes9120512

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