Recombinant Anguilla anguilla Hemoglobin anodic subunit beta (hbb1)
Description
Oxygen-Binding Properties
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Bohr Effect: Exhibits a large Bohr effect (7–8 protons bound per tetramer) linked to GTP and pH .
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Cooperative Oxygen Binding: Nonlinear Hill plots indicate T-state cooperativity, modulated by pH and organic phosphates .
Functional Divergence Between Anodic and Cathodic Hemoglobins
Eel hemoglobin isoforms exhibit specialized分工:
| Property | Anodic Hemoglobin (hbb1) | Cathodic Hemoglobin |
|---|---|---|
| Bohr Effect | Large, pH 7.5 | Reverse (negative) |
| O₂ Affinity | Low | High |
| GTP Modulation | Strong (Kd ~10⁻⁶ M) | Weak |
| Buffer Value | Low | Very Low |
| CO₂ Transport Role | Primary (67% of total Hb) | Secondary |
Genomic and Evolutionary Context
The hbb1 gene resides within the LA globin cluster (lcmt1–aqp8 chromosomal region), a product of teleost-specific whole-genome duplication (TGD) . Key evolutionary insights:
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Gene Duplication: Ancestral globin clusters (MN and LA) diverged ~320 Mya, enabling functional specialization .
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Regulatory Adaptations: Hbb1 expression correlates with hypoxia-responsive enhancers absent in cathodic globins .
Applications and Research Implications
While recombinant hbb1 production remains undocumented, native hbb1 studies inform:
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Allosteric Drug Design: Targeting β-subunit switch regions (e.g., α₁β₂ interface) for pH-stable oxygen carriers .
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Comparative Immunology: Hb-derived peptides from hbb1 show LPS-binding and peroxidase activity .
Knowledge Gaps and Future Directions
Product Specs
Target Background
Q&A
What are the distinctive features of Anguilla anguilla hemoglobin compared to other vertebrate hemoglobins?
Anguilla anguilla (European eel) hemoglobin displays several unique characteristics that distinguish it from mammalian hemoglobins. The cathodic hemoglobin of the eel plays a crucial role in oxygen transport under hypoxic and acidotic conditions. In the absence of phosphates, this hemoglobin exhibits a reverse Bohr effect and high oxygen affinity, which is strongly modulated over a wide pH range by guanosine triphosphate (GTP). The concentration of GTP in red blood cells varies with ambient oxygen availability, allowing for adaptive oxygen transport .
Complete amino acid sequence analysis of the alpha and beta chains reveals several substitutions in crucial positions compared to other hemoglobins. These include the replacement of the C-terminal His of the beta chain with Phe (which suppresses the alkaline Bohr effect) and changes in residues at the switch region between alpha and beta subunits that may alter the allosteric equilibrium .
What expression systems have proven effective for recombinant fish hemoglobins?
Recombinant hemoglobins can be heterologously expressed in various systems including:
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Transgenic bacteria (particularly E. coli)
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Yeast
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Mammalian cells (including CHO cells)
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Transgenic mice and swine
For fish hemoglobins specifically, bacterial expression systems are often preferred due to their simplicity and high yield. E. coli expression strains like Transetta (DE3) have been successfully used for recombinant hemoglobin production . The choice of expression system depends on research requirements such as post-translational modifications or protein yield.
What are the fundamental challenges in expressing functional fish hemoglobin?
Expression of functional fish hemoglobin presents several challenges:
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Ensuring proper folding and assembly of hemoglobin tetramers
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Incorporating heme groups correctly
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Overcoming the potentially low intrinsic solubility of some fish hemoglobins
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Achieving efficient post-translational modifications
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Preventing protein aggregation during expression
These challenges may be addressed by optimizing expression conditions, including induction temperature, induction time, and careful selection of E. coli expression strains .
How does the amino acid sequence of hemoglobin beta subunits influence function in fish species?
Fish hemoglobin beta subunits contain conserved residues involved in organic phosphate binding in the beta cleft, which contribute to the reverse Bohr effect in the absence of alkaline Bohr groups. In Anguilla anguilla, the replacement of the C-terminal His of the beta chain with Phe suppresses the alkaline Bohr effect. Additionally, His beta 143, which is considered responsible for the reverse Bohr effect in human and tadpole hemoglobins, is replaced by Lys in eel hemoglobin .
These sequence variations result in altered oxygen-binding properties, contributing to the hemoglobin's high intrinsic oxygen affinity and low cooperativity in the absence of allosteric effectors .
What expression conditions optimize recombinant fish hemoglobin production in E. coli?
Optimal expression of recombinant fish hemoglobins in E. coli typically involves the following conditions:
| Parameter | Recommended Conditions | Rationale |
|---|---|---|
| Growth temperature | 37°C until induction, then 28°C | Lower temperature after induction enhances protein solubility |
| Media | Terrific Broth (TB) or 2xYT | Rich media supports higher cell density and protein yield |
| Cell density at induction | OD600 of 0.6-0.8 | Optimal cell density for protein expression |
| IPTG concentration | 0.2 mM | Moderate inducer concentration prevents protein aggregation |
| Supplements | Hemin (50 μg/ml) and glucose (20 g/L) | Hemin improves heme incorporation; glucose provides energy |
| Expression time | 16 hours | Extended time allows for proper folding and assembly |
| Shaking speed | 200 rpm | Ensures proper aeration during expression |
This protocol has been effective for expressing hemoglobins with varying solubilities from different species .
What purification strategies are most effective for recombinant fish hemoglobins?
Effective purification strategies for recombinant fish hemoglobins include:
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Affinity chromatography using HisTrap FF columns for His-tagged proteins
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Purification under denaturing conditions for hemoglobins with low solubility
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Protein refolding by urea gradient dialysis at 4°C
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Size-exclusion chromatography to separate properly folded tetramers from aggregates
For instance, recombinant hemoglobins from the blood clam Tegillarca granosa were successfully purified using affinity chromatography under denaturing conditions followed by refolding through urea gradient dialysis . Similar approaches can be adapted for Anguilla anguilla hemoglobin purification.
How can researchers verify proper folding and function of recombinant fish hemoglobin?
Verification of proper folding and function should include multiple complementary approaches:
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Spectroscopic analysis: UV-visible spectroscopy to confirm proper heme incorporation and oxidation state
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Circular dichroism: To assess secondary structure elements
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Oxygen-binding assays: Measurement of oxygen affinity (P50) and cooperativity (Hill coefficient)
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Analysis of the Bohr effect: Evaluating the effect of pH on oxygen binding
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Response to allosteric effectors: Testing the modulation of oxygen affinity by GTP or other phosphates
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Thermal stability assessment: Differential scanning calorimetry to assess protein stability
These analyses collectively provide a comprehensive assessment of the structural integrity and functional properties of the recombinant hemoglobin.
What approaches can enhance the solubility of recombinant fish hemoglobin during expression?
To enhance solubility of recombinant fish hemoglobin during expression:
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Optimize induction temperature: Lower temperatures (28°C) generally favor soluble protein production
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Test multiple E. coli expression strains: Different strains have varying capacities for proper protein folding
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Adjust induction time and inducer concentration: Extended expression periods with lower IPTG levels can improve solubility
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Supplement with heme precursors: Addition of δ-aminolevulinic acid or hemin to the culture medium
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Co-express with molecular chaperones: Though not always necessary, chaperones can assist proper folding
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Use fusion partners: Solubility-enhancing protein tags (e.g., MBP, SUMO) can improve expression
These approaches can be systematically tested to identify optimal conditions for specific hemoglobin variants .
What molecular mechanisms underlie the reverse Bohr effect in Anguilla anguilla hemoglobin?
The reverse Bohr effect in Anguilla anguilla hemoglobin (increased oxygen affinity with decreasing pH) involves complex molecular mechanisms:
The conserved residues binding organic phosphate in the beta cleft likely contribute to this phenomenon. In the absence of alkaline Bohr groups, these residues may influence proton binding and subsequent conformational changes in a manner opposite to the classical Bohr effect .
The replacement of His beta 143 (responsible for the reverse Bohr effect in human and tadpole hemoglobins) with Lys in eel hemoglobin suggests species-specific mechanisms. This substitution likely alters the pKa values of key residues involved in pH-dependent conformational changes .
GTP binding obliterates the reverse Bohr effects in the cathodic hemoglobin, indicating that allosteric effectors dramatically modify the protein's response to pH changes. This complex interplay between pH and allosteric effectors allows for sophisticated regulation of oxygen binding under varying environmental conditions .
How do structural differences in fish hemoglobin beta subunits contribute to functional adaptations?
Structural differences in fish hemoglobin beta subunits contribute to functional adaptations in several ways:
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Alterations in the switch region between alpha and beta subunits affect the allosteric equilibrium between relaxed (R) and tense (T) states, modifying intrinsic oxygen affinity and cooperativity
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Substitutions at key phosphate-binding sites modify the response to allosteric effectors like GTP
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Changes in residues involved in subunit interfaces influence tetramer stability and the transmission of conformational changes
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Modifications in surface residues may affect interactions with red blood cell components
These adaptations collectively allow fish hemoglobins to function optimally in diverse aquatic environments with varying oxygen levels, temperatures, and pH conditions .
What approaches can be used to study hemoglobin allostery in recombinant fish hemoglobin systems?
Several sophisticated approaches can be employed to study hemoglobin allostery:
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Site-directed mutagenesis to test the role of specific residues in allosteric transitions
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Hydrogen-deuterium exchange mass spectrometry to identify regions undergoing conformational changes
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Time-resolved X-ray crystallography to capture transient allosteric intermediates
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Molecular dynamics simulations to model the propagation of allosteric signals through the protein
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Resonance Raman spectroscopy to study heme-protein interactions during oxygen binding
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Nuclear magnetic resonance (NMR) to detect structural changes upon ligand binding
These techniques, applied to recombinant fish hemoglobin, can reveal the molecular details of allostery and how it differs from mammalian systems .
How might directed evolution approaches be applied to engineer fish hemoglobin with enhanced properties?
Directed evolution approaches for engineering fish hemoglobin could include:
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Development of high-throughput screening systems for oxygen binding properties
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Creation of mutant libraries through error-prone PCR or DNA shuffling
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Selection strategies based on growth advantage in oxygen-limited conditions
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Iterative rounds of selection and diversification to optimize specific properties
These approaches could potentially advance the field more rapidly if appropriate screening methodologies are developed. Such engineered hemoglobins might have enhanced oxygen delivery properties, increased stability, or novel functions for biotechnological applications .
How should researchers analyze and interpret oxygen binding data from recombinant fish hemoglobin?
Analysis and interpretation of oxygen binding data should include:
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Determination of P50 (oxygen pressure at 50% saturation) under standardized conditions
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Calculation of the Hill coefficient (n) to quantify cooperativity
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Construction of complete oxygen equilibrium curves at multiple pH values to characterize the Bohr effect
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Testing with physiologically relevant concentrations of allosteric effectors (GTP, ATP)
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Comparison with native hemoglobin when possible to validate recombinant protein function
When analyzing contradictory results, researchers should carefully consider differences in experimental conditions, protein preparation methods, or the presence of undetected impurities. Systematic comparisons under standardized conditions are essential for resolving discrepancies .
What are the potential applications of recombinant Anguilla anguilla hemoglobin in research and biotechnology?
Recombinant Anguilla anguilla hemoglobin has several potential applications:
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As a model system for studying adaptations to hypoxic environments
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For developing hemoglobin-based oxygen carriers with specialized properties
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In comparative studies of protein evolution and environmental adaptation
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For investigating structure-function relationships in allosteric proteins
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As a potential antimicrobial agent, given that some fish hemoglobins have demonstrated antibacterial activity
The unique properties of eel hemoglobin, such as the reverse Bohr effect and its response to allosteric effectors, make it particularly interesting for specialized oxygen delivery applications .
How do post-translational modifications affect recombinant fish hemoglobin function?
Post-translational modifications can significantly impact recombinant fish hemoglobin function:
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Proper heme incorporation is essential for oxygen binding
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Oxidation of the heme iron affects oxygen affinity and can convert functional hemoglobin to non-functional methemoglobin
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Protein glycation or oxidative modifications can alter stability and function
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Disulfide bond formation influences protein structure and subunit interactions
When expressing recombinant fish hemoglobin, researchers must carefully assess these modifications to ensure that the recombinant protein accurately represents the native state .
What bioinformatic approaches yield the most insight into fish hemoglobin evolution and function?
Productive bioinformatic approaches include:
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Phylogenetic analysis to reconstruct the evolutionary history of fish hemoglobin genes
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Comparative genomics to identify patterns of gene duplication and subfunctionalization
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Molecular evolutionary analyses to detect signatures of selection pressure
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Homology modeling based on available crystal structures
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Molecular dynamics simulations to predict the effects of amino acid substitutions
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Network analysis of co-evolving residues to identify functionally linked positions
These approaches can provide insights into how fish hemoglobins have adapted to different environmental conditions and help identify functionally important residues that may be targets for mutagenesis studies .
What emerging technologies might advance our understanding of fish hemoglobin structure and function?
Several emerging technologies hold promise for advancing fish hemoglobin research:
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Cryo-electron microscopy for high-resolution structural studies without crystallization
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Single-molecule techniques to observe conformational changes during oxygen binding
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Advanced mass spectrometry methods for detecting subtle structural changes
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Microfluidic systems for high-throughput functional analysis
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AI-based protein structure prediction tools like AlphaFold2 for modeling fish hemoglobin variants
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Gene editing technologies for creating model organisms with modified hemoglobins
These technologies could provide unprecedented insights into hemoglobin dynamics and function .
How might comparative studies across fish species inform hemoglobin engineering?
Comparative studies across fish species living in diverse environments can:
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Identify natural solutions to specific functional challenges
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Reveal convergent evolutionary strategies for similar environmental pressures
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Discover unique structural adaptations that could be incorporated into engineered hemoglobins
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Provide a library of natural variants with specialized properties
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Establish correlation between specific amino acid substitutions and functional adaptations
This knowledge could inform rational design of hemoglobins with tailored properties for biotechnological applications or basic research .
What strategies might overcome the challenges of expressing complex hemoglobin tetramers?
Innovative strategies to overcome expression challenges include:
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Co-expression of alpha and beta chains from a single plasmid with optimal stoichiometry
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Creation of genetically linked hemoglobin subunits to ensure proper assembly
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Development of specialized E. coli strains optimized for hemoglobin expression
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Use of cell-free protein synthesis systems with controlled redox environments
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Incorporation of stabilizing mutations identified through comparative analysis
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Experimental evolution of expression hosts for improved hemoglobin production
These approaches could significantly improve the yield and quality of recombinant fish hemoglobin tetramers .
How might the unique properties of fish hemoglobin contribute to the development of novel oxygen carriers?
The unique properties of fish hemoglobin could inspire novel oxygen carriers with:
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Optimized oxygen affinity for specific medical applications
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Modified responses to pH to enhance oxygen delivery to hypoxic tissues
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Controlled rates of oxygen association and dissociation
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Reduced nitric oxide scavenging to minimize vasoconstriction
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Enhanced stability in circulation without the need for extensive chemical modification
Fish hemoglobins adapted to function in extreme environments provide natural templates for designing oxygen carriers with specialized properties. The reverse Bohr effect observed in Anguilla anguilla hemoglobin, for instance, could be particularly valuable for developing oxygen carriers that preferentially release oxygen in acidic environments such as hypoxic tumors .
