Recombinant Aldabrachelys elephantina Hemoglobin A subunit alpha-1

What is Aldabrachelys elephantina Hemoglobin A subunit alpha-1 and how does it differ from other hemoglobins?

Aldabrachelys elephantina (Aldabra giant tortoise) Hemoglobin A subunit alpha-1 is one of the globin protein chains that constitute the functional hemoglobin molecule in this reptile species. The hemoglobin of Aldabrachelys elephantina contains distinct alpha and beta subunits similar to human hemoglobin, but with significant sequence variations reflecting evolutionary adaptation to the species' unique physiology and environment .

Structurally, Aldabrachelys hemoglobin maintains the characteristic tertiary structure of vertebrate hemoglobins with a heme group that binds oxygen, but exhibits unique amino acid substitutions that may affect oxygen affinity, cooperativity, and response to allosteric regulators. Available sequence data shows that the alpha-2 subunit consists of 142 amino acids (mature protein form), while the beta subunit (specifically the A/D subunit beta) contains 146 amino acids .

What are the optimal storage and handling conditions for recombinant Aldabrachelys hemoglobin proteins?

For optimal preservation of recombinant Aldabrachelys hemoglobin proteins:

• Store at -20°C for regular use; for extended storage, maintain at -80°C

• Avoid repeated freeze-thaw cycles as this significantly reduces protein stability and activity

• When working with the protein, store aliquots at 4°C for up to one week

• Prior to use, briefly centrifuge vials to bring contents to the bottom

• Reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• For long-term storage of reconstituted protein, add glycerol to a final concentration of 5-50% (recommended 50%) and aliquot before freezing

The shelf life of liquid preparations is approximately 6 months at -20°C/-80°C, while lyophilized forms remain stable for up to 12 months at these temperatures.

What expression systems are most effective for producing recombinant Aldabrachelys hemoglobin?

Two primary expression systems have been successfully used for producing recombinant Aldabrachelys hemoglobin:

E. coli expression system:

• Advantages: High yield potential, cost-effective, well-established protocols

• Challenges: Lacks post-translational modifications, potential for inclusion body formation

• Optimization techniques: Lowering induction temperature (25-30°C), using specialized E. coli strains optimized for heterologous protein expression

• Example: The CSB-EP307887AGT product uses E. coli for expression of the beta subunit

Mammalian cell expression system:

• Advantages: Better post-translational modifications, potentially improved folding

• Challenges: Higher cost, potentially lower yields

• Example: The CSB-MP307887AGT product uses mammalian cell expression for the beta subunit

Research indicates that expression conditions significantly impact yield and solubility. For challenging hemoglobins with low intrinsic solubility, optimization of induction temperature, induction time, and E. coli strain selection can dramatically improve outcomes .

What are the key methodological considerations for purifying recombinant tortoise hemoglobin?

A successful purification strategy for recombinant tortoise hemoglobin typically involves:

• Initial capture step:

  • Ion exchange chromatography using CaptoS resin (GE Healthcare) or TREN resin (Bio-Works)

  • Buffer optimization based on the protein's isoelectric point (pI)

• Polishing step:

  • Q HP resin for final purification

  • Size exclusion chromatography to ensure homogeneity

• Quality control checks:

  • SDS-PAGE analysis to verify purity (target >85% purity)

  • Spectroscopic analysis to confirm proper heme incorporation

  • Mass spectrometry to verify accurate protein sequence and post-translational modifications

Optimizing these parameters has yielded reported purities of >85% for commercially available recombinant Aldabrachelys hemoglobin products .

How can researchers assess the oxygen-binding properties of recombinant Aldabrachelys hemoglobin?

To characterize the oxygen-binding properties of recombinant Aldabrachelys hemoglobin, researchers should consider:

Oxygen equilibrium curve analysis:

• Use specialized tonometry techniques to measure oxygen binding at different oxygen tensions

• Calculate P50 (oxygen tension at 50% saturation) and Hill coefficient (cooperativity)

• Compare results with human hemoglobin under identical conditions

• Examine the effects of temperature, pH, and allosteric effectors (e.g., 2,3-DPG, chloride)

Spectroscopic analysis:

• UV-visible spectroscopy to monitor conformational changes during oxygenation

• Stopped-flow spectroscopy to determine kinetics of oxygen binding and release

• Resonance Raman spectroscopy to probe heme pocket structure and dynamics

Thermodynamic analysis:

• Isothermal titration calorimetry to determine binding enthalpies

• van't Hoff analysis of temperature dependence to separate enthalpic and entropic contributions

These approaches allow for comprehensive characterization of the unique functional properties of tortoise hemoglobins compared to other species .

What methods are recommended for analyzing the oxidative stability of Aldabrachelys hemoglobin?

To assess oxidative stability and redox properties of Aldabrachelys hemoglobin, researchers should implement:

Autoxidation rate determination:

• Monitor the spontaneous oxidation of oxyHb (Fe²⁺) to metHb (Fe³⁺) spectrophotometrically

• Compare rates under varying temperature, pH, and buffer conditions

• Calculate half-times of autoxidation and compare to human hemoglobin

Ferryl (Fe⁴⁺) formation and reduction kinetics:

• Generate ferryl hemoglobin by incubating ferric hemoglobin (5 μM) with H₂O₂ (20x excess)

• Remove excess H₂O₂ with catalase (12 U)

• Add varying concentrations of ascorbic acid (0-500 μM)

• Monitor spectral changes in the 450-700 nm range

• Analyze time courses (545-630 nm) using double exponential equations

• Plot rates against ascorbic acid concentration and fit with appropriate models

DNA cleavage assay:

• Incubate supercoiled plasmid DNA with various concentrations of hemoglobin

• Monitor the formation of nicked (open circular) and linear DNA forms

• Quantify the decay of supercoiled DNA over time

• Calculate the DNA cleavage rate constant as a measure of oxidative reactivity

Note: Exact values would require specific testing of Aldabrachelys hemoglobin; these ranges are based on similar studies with other hemoglobins .

How can thermal stability of Aldabrachelys hemoglobin be assessed and optimized?

Differential Scanning Fluorimetry (DSF) methodology:

• Monitor the fluorescence ratio (350/330 nm) to detect changes in tryptophan and tyrosine exposure during unfolding

• Apply three different temperature ramps to thoroughly assess unfolding transitions

• Compare different ligand-bound states (oxy, deoxy, CO-bound) to determine their effects on stability

• Analyze unfolding curves to determine onset temperatures and transition temperatures

• Correlate thermal stability with functional properties

Research on similar hemoglobins shows that CO-bound samples typically exhibit two distinct transition temperatures, while O₂-bound samples display a single well-defined transition temperature. The first transition of CO-bound samples generally overlaps with the peak of O₂-bound samples .

What strategies can be employed to modify Aldabrachelys hemoglobin for enhanced properties?

Researchers can implement several approaches to modify tortoise hemoglobin:

Surface charge modification strategy:

• Introduce surface mutations to alter isoelectric point (pI)

• Target residues distant from functional regions to maintain activity

• Use site-directed mutagenesis to substitute positive charges for negative ones

• Verify changes using isoelectric focusing

• Example outcome: Surface charge mutations can lower pI from ~7.1 to ~5.8

Oxidative stability enhancement:

• Introduce tyrosine residues on the protein surface to create electron transport pathways

• Target positions like L96Y which can enhance ascorbate's ability to reduce ferryl heme

• Validate using DNA cleavage assays and lipid peroxidation measurements

Expression optimization:

• Introduce N-terminal deletions to facilitate production in E. coli

• Consider co-expression with molecular chaperones for problematic variants

• Engineer specialized fusion tags that can be removed post-purification

Verification of modifications:

• Use size exclusion chromatography with and without haptoglobin to verify that modifications don't disrupt important protein-protein interactions

• Perform comprehensive functional assays to ensure activity is maintained

How does Aldabrachelys hemoglobin contribute to our understanding of hemoglobin evolution?

Studying Aldabrachelys hemoglobin provides valuable insights into hemoglobin evolution:

• Phylogenetic analysis: The Aldabra giant tortoise (Aldabrachelys gigantea, previously known as Geochelone gigantea) represents an ancient reptilian lineage whose hemoglobin structure can reveal evolutionary adaptations specific to chelonians . Comparing the amino acid sequences of its hemoglobin with those from other vertebrates helps reconstruct the evolutionary history of globin genes.

• Functional adaptations: Unique amino acid substitutions in tortoise hemoglobin may reflect adaptations to:

  • Low metabolic rates characteristic of tortoises

  • Ability to tolerate periods of hypoxia

  • Temperature fluctuations in their environment

• Comparative analysis methodology:

  • Align alpha and beta chain sequences across species using multiple sequence alignment tools

  • Identify conserved versus variable regions

  • Calculate rates of nonsynonymous to synonymous substitutions

  • Map substitutions onto 3D structures to determine their functional significance

• Hemoglobin switching insights: While human hemoglobin undergoes developmental switching (HbF to HbA), understanding the mechanisms and evolution of this process requires comparative studies across diverse species . Reptilian hemoglobins provide an important evolutionary reference point.

What can the A/D hybrid hemoglobin of Aldabrachelys tell us about globin gene evolution?

The presence of A/D hybrid hemoglobin in the Aldabra giant tortoise offers unique perspectives on globin gene evolution:

• Globin gene family history: The A/D hybrid hemoglobin in Aldabrachelys represents an interesting case study in globin gene evolution. While mammals typically have distinct alpha and beta globin clusters, reptiles often show more complex arrangements with hybrid forms.

• Methodological approach to evolutionary analysis:

  • Compare A/D hybrid sequences with conventional alpha and beta globins

  • Analyze conserved functional regions versus variable segments

  • Examine intron-exon structure of globin genes

  • Reconstruct the evolutionary history of gene duplication and divergence events

• Functional implications: The A/D hybrid may confer specific oxygen-binding properties advantageous for the tortoise's physiology and ecology. Studying these functional properties can reveal how structural variations translate to adaptive advantages.

• Research applications: The unique properties of A/D hybrid hemoglobin could inform the design of hemoglobin-based oxygen carriers (HBOCs) by revealing novel structure-function relationships .

How can recombinant Aldabrachelys hemoglobin be used in studies of oxidative stress mechanisms?

Recombinant Aldabrachelys hemoglobin offers a valuable tool for investigating oxidative stress mechanisms:

• Comparative oxidative reactivity studies:

  • Compare the peroxidase-like activity of tortoise versus mammalian hemoglobins

  • Measure reactions with hydrogen peroxide to form ferryl species

  • Quantify rates of reaction with various substrates

  • Assess damage to lipids, proteins, and DNA

• Experimental design for oxidative stress studies:

  • Lipoprotein peroxidation assay: Incubate hemoglobin with reconstituted lipoproteins and measure formation of conjugated dienes spectrophotometrically

  • Protein oxidation analysis: Quantify formation of protein carbonyls and advanced oxidation protein products

  • Cellular models: Expose cultured cells to hemoglobin under various conditions and assess viability, redox status, and antioxidant responses

• Technical considerations:

  • Maintain hemoglobin in defined redox states (oxy, deoxy, met) for experiments

  • Control for potential contaminants like catalase or superoxide dismutase

  • Use multiple complementary assays to comprehensively characterize oxidative processes

• Research applications:

  • Understanding species-specific differences in hemoglobin oxidative stability

  • Developing improved hemoglobin-based oxygen carriers with reduced oxidative toxicity

  • Elucidating evolutionary adaptations in antioxidant mechanisms

How can researchers address challenges in studying the redox properties of tortoise hemoglobin?

Studying the redox properties of tortoise hemoglobin presents several methodological challenges that researchers must address:

• Challenge: Maintaining defined redox states

  • Solution: Develop standardized protocols for preparing oxy-, deoxy-, and met-hemoglobin forms:

    • For oxyHb: Expose hemoglobin to oxygen-saturated buffer and verify >98% oxygenation spectrophotometrically

    • For metHb: Oxidize with slight excess of ferricyanide and remove excess oxidant by gel filtration

    • For deoxyHb: Use vacuum or nitrogen purging followed by addition of sodium dithionite

• Challenge: Accurately measuring fast reaction kinetics

  • Solution: Employ stopped-flow spectroscopy with rapid mixing capabilities:

    • For autoxidation studies: Monitor absorbance changes at 576 nm (oxy) and 630 nm (met)

    • For NO reactions: Use specialized NO-delivery systems with precise concentration control

    • For peroxide reactions: Implement rapid-scan capabilities to capture transient intermediates

• Challenge: Distinguishing subunit-specific behaviors

  • Solution: Use specialized spectroscopic techniques and hybrid hemoglobins:

    • Apply second-derivative spectroscopy to resolve overlapping spectral features

    • Create hybrid hemoglobins combining human and tortoise subunits to isolate subunit properties

    • Analyze time courses with multi-exponential fits to resolve subunit contributions

• Challenge: Accounting for temperature effects

  • Solution: Implement careful temperature control and comparative analyses:

    • Perform studies at both standard temperature (25°C) and physiologically relevant reptilian temperatures

    • Develop van't Hoff plots to extract thermodynamic parameters

    • Compare temperature effects on tortoise hemoglobin versus mammalian hemoglobins

By addressing these methodological challenges, researchers can generate reliable data on the unique redox properties of tortoise hemoglobin and their functional implications .

What are the most promising applications of Aldabrachelys hemoglobin research for hemoglobin-based oxygen carriers?

The unique properties of Aldabrachelys hemoglobin offer several promising avenues for hemoglobin-based oxygen carrier (HBOC) research:

• Oxidative stability enhancement:

  • Tortoise hemoglobins may exhibit naturally evolved resistance to oxidative damage

  • Identify specific amino acid residues responsible for enhanced stability

  • Incorporate these features into next-generation HBOCs through site-directed mutagenesis

• Novel crosslinking approaches:

  • Study the surface topography of tortoise hemoglobin to identify optimal crosslinking sites

  • Develop specialized crosslinkers that preserve the functional properties of the protein

  • Create genetically crosslinked tetramers similar to di-α constructs used in human HBOCs

• PEGylation strategies:

  • Investigate PEGylation of tortoise hemoglobin using the Euro-PEG-Hb method

  • Compare vascular retention and functional properties with PEGylated human hemoglobin

  • Optimize PEG size and attachment sites for optimal performance

• Hybrid hemoglobin designs:

  • Create chimeric proteins combining advantageous features from human and tortoise hemoglobins

  • Incorporate electron transfer pathways from surface tyrosines observed in some hemoglobin variants

  • Engineer hemoglobins with reduced NO scavenging through strategic mutations

• Experimental validation approaches:

  • Oxidative stress models: Test HBOC candidates in standardized assays of lipid peroxidation

  • Animal models: Evaluate performance in hemorrhagic shock and ischemia-reperfusion models

  • Analytical techniques: Develop specialized mass spectrometry approaches to characterize oxidative modifications

How can comparative studies between human and tortoise hemoglobin inform therapeutic hemoglobin design?

Comparative studies between human and tortoise hemoglobin can significantly advance therapeutic hemoglobin design:

• Structure-function relationship analysis:

  • Identify key amino acid differences in regions affecting:

    • Oxygen affinity and cooperativity

    • Redox stability and autoxidation rates

    • Sensitivity to allosteric effectors

  • Use site-directed mutagenesis to incorporate beneficial tortoise hemoglobin features into human hemoglobin templates

• Oxidative damage resistance mechanisms:

  • Compare rates of ferryl formation and reduction between species

  • Identify natural amino acids acting as redox-active sites in tortoise hemoglobin

  • Map electron transfer pathways that may protect against oxidative damage

  • Example methodology: Monitor ferryl reduction using time-resolved spectroscopy with ascorbate as reducing agent

• Surface property optimization approach:

  • Compare surface charge distributions and their effects on:

    • Protein-protein interactions

    • Vascular retention

    • Interaction with scavenging proteins like haptoglobin

  • Design modified hemoglobins with optimized surface properties based on insights from tortoise hemoglobin

• Experimental design for comparative assessment:

  • Standardized oxidative challenge assays

  • Tissue perfusion studies comparing oxygen delivery efficiency

  • Nitric oxide reactivity measurements to assess vasoactivity risk

This comparative approach leverages evolutionary adaptations that have been refined over millions of years to inform rational protein engineering for therapeutic applications .