Recombinant Chlorocebus aethiops Hemoglobin subunit alpha (HBA)

What is Chlorocebus aethiops Hemoglobin subunit alpha and how does it compare to human HBA?

Chlorocebus aethiops Hemoglobin subunit alpha (HBA) is the alpha chain component of hemoglobin in African green monkeys. Similar to human HBA, it functions primarily in oxygen transport from the lungs to peripheral tissues. Both proteins belong to the globin family and share high sequence homology, though species-specific amino acid variations exist that may influence oxygen binding affinity and other functional properties. The protein also possesses secondary functions; for example, hemopressin derived from hemoglobin acts as an antagonist peptide of the cannabinoid receptor CNR1, efficiently blocking it and subsequent signaling pathways .

What are the typical hematological parameters for Chlorocebus aethiops and how do they differ from human values?

Chlorocebus aethiops (African green monkeys) display hematological parameters that share similarities with humans but with species-specific ranges. Normal adult values include:

These values are particularly relevant when using Chlorocebus aethiops as a model for human diseases or when evaluating the physiological effects of recombinant HBA administration .

What expression systems are typically used for recombinant Chlorocebus aethiops HBA production?

While the search results don't specifically address expression systems for Chlorocebus aethiops HBA, insights can be drawn from recombinant human hemoglobin production. Recombinant hemoglobin is commonly expressed in bacterial systems like Escherichia coli, which require modifications such as the V1M mutation to facilitate proper expression. Plant-based expression systems, such as wheat germ, have also proven effective for producing full-length hemoglobin subunits suitable for analysis by techniques like ELISA and Western blotting .

For optimal expression, the initiator methionine must be properly processed, as it may not be cleaved in certain variants (like variant Thionville) and can be acetylated . Some recombinant hemoglobin designs incorporate a fused di-α gene inserted in an operon with the β-gene to express a tetramer that resists dissociation into α₁β₁ dimers under physiological conditions .

How should researchers design experiments to evaluate oxygen binding properties of recombinant Chlorocebus aethiops HBA?

When designing experiments to evaluate oxygen binding properties of recombinant Chlorocebus aethiops HBA, researchers should:

• Purification protocol: Utilize affinity chromatography followed by size-exclusion chromatography to ensure high purity of the recombinant protein.

• Spectroscopic analysis: Implement UV-visible spectroscopy to characterize the heme environment and confirm proper folding of the recombinant protein.

• Oxygen equilibrium curves: Generate oxygen equilibrium curves under varying conditions (pH, temperature, presence of allosteric effectors) to determine:

  • P₅₀ values (oxygen pressure at 50% saturation)

  • Hill coefficients (cooperativity)

  • Bohr effect (pH-dependent oxygen affinity shifts)

• Kinetic measurements: Assess association and dissociation rates of oxygen binding using stopped-flow techniques to understand the dynamic aspects of oxygen transport.

• Comparative analysis: Include human HBA as a reference standard in all experiments to contextualize findings within primate hemoglobin evolution .

What modifications can be introduced to optimize recombinant Chlorocebus aethiops HBA for specific research applications?

Several strategic modifications can be introduced to optimize recombinant Chlorocebus aethiops HBA for specific research applications:

• Oxygen affinity modulation: Targeted mutations at allosteric sites (e.g., αV96W or βK82D) can selectively stabilize the low-affinity (T) quaternary state, reducing oxygen affinity by up to 50-fold .

• Stability enhancement: Introducing mutations such as βG16A, βH116I, and αG15A can increase resistance of apohemoglobin to unfolding, improving the protein's stability during experimental procedures .

• Radical species protection: Replacing specific tyrosines with phenylalanines (or vice versa) and removing cysteines can decrease the lifetimes of destructive protein radicals and ferryl species after treatment with H₂O₂, protecting the protein during oxidative stress experiments .

• Hemin retention improvement: Mutations like βS44H can decrease the rate of hemin dissociation from β-subunits, preventing the loss of the crucial prosthetic group during extended experiments .

• Nitric oxide dioxygenation (NOD) control: Strategic substitutions at B10, E11, and G8 positions can modulate the rates of the NOD reaction while maintaining oxygen binding capabilities, which is particularly valuable for studies investigating hemoglobin's impact on nitric oxide signaling .

What analytical techniques are most effective for characterizing recombinant Chlorocebus aethiops HBA?

For comprehensive characterization of recombinant Chlorocebus aethiops HBA, the following analytical techniques are most effective:

• SDS-PAGE: For purity assessment and molecular weight confirmation. Protocols typically use 12.5% gels with Coomassie Blue staining, as demonstrated with recombinant human hemoglobin .

• Western blotting: For specific detection using anti-hemoglobin antibodies, confirming identity and integrity of the recombinant protein .

• ELISA: For quantitative analysis and functional antibody detection assays .

• Mass spectrometry: For precise molecular weight determination, post-translational modification mapping, and confirmation of amino acid sequence.

• Circular dichroism spectroscopy: For secondary structure analysis, ensuring proper protein folding.

• X-ray crystallography or cryo-EM: For high-resolution structural analysis, particularly important when comparing structural differences between Chlorocebus aethiops HBA and human HBA.

• Functional assays: Including oxygen binding kinetics, thermal stability measurements, and analytical ultracentrifugation for quaternary structure analysis .

How should researchers interpret differences in oxygen binding parameters between recombinant and native Chlorocebus aethiops HBA?

When interpreting differences in oxygen binding parameters between recombinant and native Chlorocebus aethiops HBA, researchers should consider:

• Expression system artifacts: The expression system (bacterial, wheat germ, etc.) may introduce subtle structural changes that affect function. For instance, incomplete processing of the initiator methionine, which occurs in some variants, can impact protein structure and function .

• Post-translational modifications: Native HBA undergoes specific post-translational modifications that may be absent in recombinant versions. For example, acetylation of the initiator methionine occurs in variant Thionville and affects protein properties .

• Heme incorporation efficiency: Inefficient or improper heme incorporation in recombinant systems can alter oxygen binding parameters. Assess the heme:protein ratio spectrophotometrically to ensure proper incorporation.

• Quaternary structure stability: Recombinant hemoglobin may show different dissociation patterns compared to native tetramers. Techniques like analytical ultracentrifugation can reveal these differences.

• Statistical validation: Apply appropriate statistical tests to determine if differences fall within experimental error or represent true biological variations. The interobserver coefficient of variation (CV) should be calculated similarly to other primate studies where values typically range from 21-26% .

Researchers should systematically characterize these differences to determine whether they represent artifacts of the recombinant production process or genuine features of the protein that may have physiological significance.

What are the expected changes in Chlorocebus aethiops hemoglobin parameters during physiological states such as pregnancy?

Pregnancy induces significant changes in Chlorocebus aethiops hemoglobin parameters that researchers should account for when using these animals as models. Based on comprehensive studies:

• Decreased red blood cell parameters: During pregnancy, females show significant reductions in:

  • RBC count: Decreases from 5.88 M/uL to 5.25 M/uL (10.7% reduction)

  • Hemoglobin (Hgb): Decreases from 13.5 g/dL to 12.8 g/dL (5.2% reduction)

  • Hematocrit (Hct): Decreases from 42.5% to 39.2% (7.8% reduction)

• Altered white blood cell profile: Pregnancy induces:

  • Increased total WBC: From 6100 K/uL to 8700 K/uL (42.6% increase)

  • Elevated neutrophils: From 3354/uL to 4524/uL (34.9% increase)

  • Increased lymphocytes: From 2160/uL to 3300/uL (52.8% increase)

• Reduced platelet count: Decreases from 310 K/uL to 242 K/uL (21.9% reduction)

These physiological adaptations parallel human pregnancy changes and should be considered when interpreting experimental results from pregnant subjects or when designing studies that include females of reproductive age.

How do temperature and pH affect the functional properties of recombinant Chlorocebus aethiops HBA?

Temperature and pH significantly impact the functional properties of recombinant Chlorocebus aethiops HBA through several mechanisms:

• Bohr effect: Like other primate hemoglobins, Chlorocebus aethiops HBA exhibits the Bohr effect, where oxygen affinity decreases with decreasing pH. This physiologically critical property facilitates oxygen release in metabolically active tissues where pH is lower.

• Temperature sensitivity: Oxygen affinity decreases with increasing temperature, following van't Hoff's principle. The enthalpy change (ΔH) of oxygenation is negative, making the reaction exothermic.

• Allosteric regulation: The binding of modulators such as 2,3-bisphosphoglycerate (2,3-BPG) is pH-dependent, with stronger binding at lower pH values, further decreasing oxygen affinity.

• Protein stability: Both extremes of pH and elevated temperatures can lead to denaturation and heme loss. This risk increases with genetic modifications, as some mutations designed to alter oxygen binding properties may simultaneously reduce protein stability .

When designing experimental protocols, researchers should carefully control both pH and temperature, as even small variations can significantly affect oxygen binding measurements and other functional assays.

How can recombinant Chlorocebus aethiops HBA be engineered for use in hemoglobin-based oxygen carriers (HBOCs)?

Engineering recombinant Chlorocebus aethiops HBA for hemoglobin-based oxygen carriers (HBOCs) requires several strategic modifications to overcome the limitations of natural hemoglobin in therapeutic contexts:

• Prevention of tetramer dissociation: Implementing a di-α gene fusion with a glycine linker between the two α-polypeptides prevents dissociation into αβ dimers, which can cause nephrotoxicity. This approach, similar to that used in human rHb0.1, maintains the tetrameric structure even at dilute concentrations .

• Optimization of oxygen affinity: Introducing mutations at allosteric sites (such as αV96W or βK82D) can selectively stabilize the low-affinity T state, reducing oxygen affinity to physiologically appropriate levels for tissue oxygen delivery .

• Enhancement of oxidative stability: Replacing oxidation-prone amino acids (particularly methionines and cysteines) with more stable residues decreases susceptibility to oxidative damage. Tyrosine-to-phenylalanine substitutions can reduce destructive protein radical formation and ferryl species after H₂O₂ exposure .

• Modulation of nitric oxide interactions: Controlled mutations at distal heme pocket positions (B10, E11, and G8) can reduce the nitric oxide dioxygenation (NOD) reaction rate while maintaining appropriate oxygen binding kinetics, helping to preserve vascular tone during HBOC administration .

• Prolonged circulation time: PEGylation or encapsulation strategies can increase the half-life in circulation and reduce immune recognition and clearance.

When developing these engineered HBOCs, researchers must balance multiple, sometimes competing, biochemical properties to achieve an effective oxygen carrier with minimal side effects.

What approaches can be used to study the evolutionary adaptations in Chlorocebus aethiops HBA compared to other primate hemoglobins?

To investigate evolutionary adaptations in Chlorocebus aethiops HBA compared to other primate hemoglobins, researchers can employ multiple complementary approaches:

• Comparative sequence analysis: Align Chlorocebus aethiops HBA sequences with those from diverse primate species to identify:

  • Conserved residues indicating functional constraints

  • Species-specific substitutions suggesting adaptive evolution

  • Residues under positive selection using algorithms like PAML

• Ancestral sequence reconstruction: Computationally infer ancestral hemoglobin sequences at various nodes in the primate phylogeny to track the emergence of specific adaptations.

• Structure-function correlation: Map sequence differences onto three-dimensional structures to identify how substitutions in different primate lineages affect:

  • Heme pocket architecture

  • Subunit interfaces

  • Allosteric regulation sites

• Experimental validation: Express recombinant ancestral and extant primate HBAs to measure:

  • Oxygen binding parameters under varying conditions

  • Stability against denaturation

  • Susceptibility to oxidative damage

• Ecological correlation: Analyze how hemoglobin properties correlate with the ecological niches of different primate species, such as altitude adaptation, diving behavior, or activity patterns.

This multidisciplinary approach can reveal how natural selection has fine-tuned hemoglobin properties across primate evolution to meet diverse physiological demands.

How can imaging mass spectrometry be applied to study the distribution and modifications of Chlorocebus aethiops HBA in tissues?

Imaging mass spectrometry (IMS) offers powerful capabilities for studying the spatial distribution and post-translational modifications of Chlorocebus aethiops HBA in tissues:

• Tissue preparation protocol:

  • Tissues should be procured during necropsy and immediately flash-frozen

  • Sections should be cut at 5-10 μm thickness using a cryostat

  • Matrix application must be optimized for hemoglobin detection (sinapinic acid typically works well for proteins)

• Spatial distribution mapping:

  • IMS can visualize the heterogeneous distribution of HBA across tissue sections with resolution down to 10 μm

  • In lung tissue, this can reveal oxygen exchange microenvironments

  • In infected tissues, correlation between pathogen distribution and hemoglobin degradation products can be established

• Post-translational modification detection:

  • IMS can identify and localize specific modifications including:

    • Oxidative modifications (particularly in disease states)

    • Glycation patterns

    • Proteolytic fragments such as hemopressin

• Quantitative analysis:

  • Relative quantification of HBA across different tissue regions can be achieved

  • Comparison between healthy and diseased states can reveal pathology-specific alterations

• Multi-omics integration:

  • IMS data can be correlated with histology, immunohistochemistry, and transcriptomics

  • This integrated approach links hemoglobin distribution to gene expression patterns and cellular phenotypes

This methodology is particularly valuable for studying hemoglobin's non-canonical functions beyond oxygen transport and for investigating pathological alterations in various disease models.

What are the current limitations in recombinant Chlorocebus aethiops HBA research and potential solutions?

Current limitations in recombinant Chlorocebus aethiops HBA research include:

• Expression system optimization: The ideal expression system that maintains authentic post-translational modifications while providing high yields remains elusive. Emerging eukaryotic expression systems, including modified yeast and mammalian cell lines, offer promising alternatives to traditional E. coli and wheat germ systems .

• Heme incorporation efficiency: Achieving complete and proper heme incorporation remains challenging, affecting functional studies. Improved co-expression of heme synthesis enzymes or development of more efficient in vitro heme incorporation protocols could address this limitation.

• Authentic tetramer assembly: Formation of physiologically relevant α₂β₂ tetramers requires coordinated expression of both α and β subunits. The fused di-α gene approach has shown promise but may introduce structural constraints that affect function .

• Limited comparative data: Lack of comprehensive data comparing recombinant and native Chlorocebus aethiops HBA hampers validation efforts. Establishing standardized comparative analyses between recombinant products and hemoglobin purified from Chlorocebus aethiops blood would address this gap.

• Oxidative stability: Susceptibility to oxidative damage during expression and purification affects protein quality. Integration of site-directed mutagenesis to replace oxidation-prone residues without compromising function offers one solution pathway .

Addressing these limitations through interdisciplinary approaches will significantly advance the field and expand research applications of recombinant Chlorocebus aethiops HBA.

How can computational modeling advance our understanding of Chlorocebus aethiops HBA structure-function relationships?

Computational modeling provides powerful approaches to advance our understanding of Chlorocebus aethiops HBA structure-function relationships:

• Homology modeling and molecular dynamics:

  • Generate high-resolution structural models of Chlorocebus aethiops HBA based on crystallographic templates

  • Simulate protein dynamics to identify conformational changes during oxygen binding/release

  • Calculate free energy landscapes to understand allosteric transitions between R and T states

• Quantum mechanical calculations:

  • Model the electronic structure of the heme group and its interactions with bound oxygen

  • Predict how species-specific amino acid differences affect oxygen binding energetics

  • Simulate reaction pathways for oxidative processes that lead to hemoglobin degradation

• Systems biology integration:

  • Model hemoglobin within the broader context of erythrocyte metabolism

  • Simulate how changes in hemoglobin properties affect oxygen delivery under various physiological conditions

  • Predict emergent properties from the interaction of hemoglobin with other cellular components

• Machine learning applications:

  • Develop algorithms to predict the functional consequences of specific mutations

  • Identify patterns in hemoglobin sequence variation that correlate with physiological adaptations

  • Design optimized hemoglobin variants with tailored properties for specific research applications

These computational approaches can guide experimental design, provide mechanistic insights difficult to obtain experimentally, and accelerate the development of hemoglobin-based technologies.

What are the potential applications of Chlorocebus aethiops HBA in comparative pathophysiology studies?

Recombinant Chlorocebus aethiops HBA offers valuable applications in comparative pathophysiology studies:

• Infectious disease models: African green monkeys serve as important models for various infectious diseases. Studies using Chlorocebus aethiops have been conducted for pathogens like Burkholderia mallei, making their hemoglobin relevant for understanding host-pathogen interactions at the molecular level .

• Hemoglobinopathy research: Comparing human hemoglobinopathy mutations with equivalent substitutions in Chlorocebus aethiops HBA can reveal species-specific compensatory mechanisms that might inform therapeutic strategies.

• Oxidative stress resistance: Investigating how Chlorocebus aethiops HBA responds to oxidative stress compared to human HBA may identify protective mechanisms that could be transferred to human hemoglobin through protein engineering.

• Pregnancy-related adaptations: The documented changes in hematological parameters during pregnancy in African green monkeys (including decreased RBC, hemoglobin, and hematocrit) parallel human pregnancy adaptations, making this a valuable model for studying maternal-fetal oxygen transport .

• Aging studies: As a long-lived primate species with well-characterized aging biomarkers, Chlorocebus aethiops provides opportunities to study age-related changes in hemoglobin function and post-translational modifications that may contribute to decreased tissue oxygenation in elderly populations .

These comparative studies can provide insights into both basic biological mechanisms and potential therapeutic approaches for human diseases involving hemoglobin dysfunction or oxygen transport impairment.