Recombinant Bothrops jararaca Thrombin-like enzyme bothrombin

What are the basic structural properties of recombinant bothrombin?

Recombinant Bothrops jararaca thrombin-like enzyme (bothrombin) is composed of 232 amino acid residues with a theoretical molecular weight of 33.0 kDa. When analyzed by SDS-PAGE, bothrombin shows M(r) values of 33,000 under nonreducing conditions and 35,000 under reducing conditions . The recombinant form contains N-terminal 10xHis-tag and C-terminal Myc-tag with an expression region spanning amino acids 1-232 . The native enzyme contains three Asn-linked oligosaccharide chains, which contribute to its structural stability and function . These glycosylation patterns may differ in the recombinant protein depending on the expression system used, which can impact biological activity and stability in experimental settings.

What are the main synonyms and classification identifiers for bothrombin in scientific literature?

Researchers should be aware of multiple nomenclature variants when conducting literature searches about bothrombin. The enzyme is referred to by several synonyms including: SVTLE (Snake Venom Thrombin-Like Enzyme), Factor VIII activator, Fibrinogen-clotting enzyme, Snake venom serine protease (SVSP), and Venombin A . Its accession number is P81661, which can be used to access its sequence information in protein databases . In classification systems, bothrombin belongs to the serine protease family of snake venom enzymes and is categorized as a thrombin-like enzyme based on its functional properties, despite having structural differences from mammalian thrombin.

How can researchers effectively measure bothrombin's fibrinogen-clotting activity?

To accurately measure bothrombin's fibrinogen-clotting activity, researchers should employ comparative assays against standard NIH alpha-thrombin units. The purified enzyme demonstrates specific fibrinogen-clotting activity equivalent to 814-904 NIH alpha-thrombin units/mg . A standardized protocol involves:

• Preparing purified fibrinogen at a concentration of 2-5 mg/ml in appropriate buffer

• Preparing serial dilutions of bothrombin and a reference alpha-thrombin standard

• Adding enzyme samples to fibrinogen solution at 37°C

• Measuring clotting time and calculating relative potency

The activity should be assessed using both chromogenic substrates and fibrinogen clotting assays for comprehensive characterization. When working with recombinant bothrombin, researchers should verify enzyme purity (>90% as determined by SDS-PAGE) to ensure reliable and reproducible results . Potential interference from His and Myc tags should be considered when comparing recombinant versus native enzyme activity.

What methods should be used to investigate bothrombin's interaction with coagulation factors?

Investigating bothrombin's interactions with coagulation factors, particularly Factor VIII, requires specialized approaches due to its significantly lower activation capacity compared to alpha-thrombin (approximately 950 times less) . Researchers should employ:

• Purified factor assays using chromogenic or clotting-based endpoints

• Real-time monitoring of factor activation using fluorogenic substrates

• Surface plasmon resonance or other binding assays to determine kinetic parameters

• Activated partial thromboplastin time (aPTT) assays to assess functional impacts

When designing experiments, it's crucial to use sufficiently sensitive detection methods given bothrombin's relatively weak Factor VIII activation. Additionally, controls with specific inhibitors help differentiate bothrombin's effects from those of potential contaminating proteases. Diisopropyl fluorophosphate completely abolishes bothrombin activity, while hirudin (a specific alpha-thrombin inhibitor) has negligible effects, providing useful tools for experimental validation .

What are the optimal conditions for expressing recombinant bothrombin in E. coli systems?

Expressing functional recombinant bothrombin in E. coli requires careful optimization of several parameters. The recombinant protein is typically produced with an N-terminal 10xHis-tag and C-terminal Myc-tag to facilitate purification and detection . Key considerations include:

• Codon optimization for E. coli expression, particularly for rare codons found in snake venom genes

• Selection of appropriate E. coli strain (BL21(DE3), Rosetta, or Origami strains for disulfide bond formation)

• Temperature reduction during induction (typically 16-18°C) to enhance proper folding

• Use of specialized media formulations to maximize yield

• IPTG concentration optimization (typically 0.1-0.5 mM)

The expression region spanning amino acids 1-232 must be correctly incorporated into the expression vector . Since the native bothrombin contains three Asn-linked oligosaccharide chains , researchers should be aware that E. coli-expressed protein will lack these modifications, potentially affecting certain functional properties. Alternative expression systems such as yeast or mammalian cells may be considered if glycosylation is critical for the specific research application.

What purification strategy yields the highest purity and activity for recombinant bothrombin?

A multi-step purification strategy is recommended to achieve >90% purity for recombinant bothrombin as determined by SDS-PAGE . An effective purification protocol includes:

Throughout purification, it's essential to monitor both protein concentration and enzymatic activity to calculate specific activity and recovery rates. The final purified product should be assessed for fibrinogen-clotting activity equivalent to 814-904 NIH alpha-thrombin units/mg to confirm functional integrity . Proper storage conditions (-80°C with glycerol or lyophilized) are crucial for maintaining long-term stability.

How can researchers verify the structural integrity of purified recombinant bothrombin?

Verifying the structural integrity of purified recombinant bothrombin requires multiple analytical approaches:

• SDS-PAGE under both reducing and non-reducing conditions to confirm the expected molecular weight (33 kDa under non-reducing and 35 kDa under reducing conditions)

• Western blotting using anti-His and anti-Myc antibodies to confirm tag presence and integrity

• Mass spectrometry analysis to verify the exact molecular weight and potential post-translational modifications

• Circular dichroism spectroscopy to assess secondary structure elements

• Limited proteolysis followed by mass spectrometry to evaluate domain folding

• Activity assays measuring fibrinogen-clotting capability to confirm functional integrity

Additionally, researchers should compare the recombinant protein's properties with those of native bothrombin isolated from Bothrops jararaca venom when possible. Particular attention should be paid to the absence of glycosylation in E. coli-expressed protein, as the native bothrombin contains three Asn-linked oligosaccharide chains that may influence structure and function .

How does bothrombin's mechanism differ from other snake venom thrombin-like enzymes (SVTLEs)?

Bothrombin exhibits distinct mechanistic properties compared to other snake venom thrombin-like enzymes (SVTLEs). Unlike many SVTLEs that cleave either fibrinopeptide A or both fibrinopeptides A and B, bothrombin selectively cleaves fibrinopeptide A without releasing fibrinopeptide B . This selective action results in the formation of abnormal fibrin clots that are more susceptible to fibrinolysis. Another distinctive feature is bothrombin's interaction with platelets, which requires the presence of exogenous fibrinogen and involves binding to glycoprotein Ib (GP Ib) . This binding mechanism differs from many other SVTLEs and more closely resembles alpha-thrombin's platelet interaction, though the downstream signaling pathways diverge significantly.

The inhibitor profile of bothrombin also distinguishes it from other SVTLEs. While diisopropyl fluorophosphate completely abolishes bothrombin activity (confirming its identity as a serine protease), hirudin—a specific alpha-thrombin inhibitor—has negligible effect on bothrombin activity . This inhibition pattern can help researchers differentiate bothrombin-mediated effects from those of other coagulation enzymes in complex experimental systems.

What experimental design best highlights the functional differences between bothrombin and alpha-thrombin?

To effectively demonstrate the functional differences between bothrombin and alpha-thrombin, a comprehensive experimental design should include parallel assays addressing multiple parameters:

Using platelets from Bernard-Soulier syndrome patients (lacking GP Ib) provides a particularly insightful model, as these platelets respond to alpha-thrombin but not to bothrombin even in the presence of exogenous fibrinogen . This experimental approach directly highlights the differential receptor requirements between these enzymes.

How can bothrombin be distinguished from other coagulation enzymes in complex biological samples?

Distinguishing bothrombin from other coagulation enzymes in complex biological samples requires a strategic combination of biochemical and immunological approaches:

• Selective inhibition profiles: Bothrombin is inhibited by diisopropyl fluorophosphate but shows negligible inhibition by hirudin . This contrasts with alpha-thrombin, which is highly sensitive to hirudin.

• Substrate specificity analysis: Bothrombin's selective cleavage of fibrinopeptide A without affecting fibrinopeptide B provides a distinctive enzymatic signature . HPLC or mass spectrometry analysis of fibrinopeptide release patterns can confirm bothrombin activity.

• Immunological detection: Using specific anti-bothrombin antibodies in Western blotting or ELISA assays. For recombinant bothrombin, anti-His and anti-Myc antibodies can detect the tagged protein .

• Molecular weight verification: Bothrombin shows characteristic M(r) values of 33,000 under nonreducing and 35,000 under reducing conditions on SDS-PAGE .

• Functional bioassays: Bothrombin's unique requirement for exogenous fibrinogen to induce platelet aggregation, and the complete inhibition of this activity by anti-GP Ib antibodies, provides a functional fingerprint .

These approaches, especially when used in combination, can reliably identify bothrombin activity even in the presence of other proteases and coagulation factors.

What structural features account for bothrombin's selective cleavage of fibrinopeptide A?

The selective cleavage of fibrinopeptide A without releasing fibrinopeptide B by bothrombin is attributed to specific structural features within its active site and substrate recognition regions. Though the complete three-dimensional structure is not fully detailed in the provided search results, comparative analysis with other SVTLEs suggests several critical features:

• The substrate binding pocket of bothrombin likely contains specific residues that recognize the Arg-Gly bond in the Aα chain of fibrinogen while having reduced affinity for the corresponding region in the Bβ chain.

• The enzyme's primary structure, comprising 232 amino acid residues , includes a catalytic triad characteristic of serine proteases, but with subtle differences in the arrangement of subsites that determine substrate specificity.

• The three Asn-linked oligosaccharide chains may influence the enzyme's conformation and accessibility of its active site, potentially contributing to its selective activity.

Understanding these structural determinants requires crystallographic studies or molecular modeling based on the known amino acid sequence. These approaches would enable researchers to identify the specific residues involved in substrate recognition and potentially engineer variants with modified specificities.

What is the molecular basis for bothrombin's interaction with platelet glycoprotein Ib?

The molecular interaction between bothrombin and platelet glycoprotein Ib (GP Ib) represents a critical mechanism for bothrombin's effects on platelets. Evidence for this interaction comes from experiments with platelets from a patient with Bernard-Soulier syndrome, which lack GP Ib and do not respond to bothrombin even in the presence of exogenous fibrinogen . This suggests that GP Ib, rather than the recently cloned thrombin receptor, serves as the initial binding site for bothrombin on platelets.

The molecular determinants of this interaction likely include:

• Specific exosite regions on bothrombin that recognize GP Ib, potentially similar to but distinct from the corresponding regions in alpha-thrombin

• Charge-dependent interactions, as suggested by the inhibitory effect of EDTA (10 mM) on bothrombin-induced platelet aggregation

• Conformational requirements evidenced by the inability of bothrombin to directly activate platelets without fibrinogen binding to GP IIb/IIIa

This complex interplay between bothrombin, fibrinogen, GP Ib, and GP IIb/IIIa suggests a sequential activation mechanism that differs from the direct thrombin receptor activation by alpha-thrombin. Detailed structural studies using techniques such as co-crystallization or molecular docking would provide further insights into this unique interaction.

How do the three Asn-linked oligosaccharide chains affect bothrombin's function and stability?

The native bothrombin contains three Asn-linked oligosaccharide chains that likely play multiple roles in the enzyme's function and stability:

• Structural stability: The glycans may contribute to proper folding and resistance to proteolytic degradation, enhancing the enzyme's half-life in circulation.

• Solubility and aggregation resistance: Oligosaccharides increase protein hydrophilicity and reduce aggregation potential, particularly important for a protein that functions in aqueous plasma environments.

• Recognition and clearance: Glycosylation patterns can influence how the protein interacts with cellular receptors and clearance mechanisms in vivo.

• Enzymatic activity modulation: The positioning of glycans relative to the active site may influence substrate accessibility and catalytic efficiency.

Researchers working with recombinant bothrombin expressed in E. coli should note that this expression system does not perform N-linked glycosylation , resulting in a non-glycosylated protein that may exhibit differences in stability, activity, or interaction patterns compared to the native enzyme. Comparative studies between glycosylated (from natural sources or eukaryotic expression systems) and non-glycosylated forms would provide valuable insights into the functional significance of these post-translational modifications.

How can researchers address activity loss during purification and storage of recombinant bothrombin?

Activity loss during purification and storage represents a common challenge when working with recombinant bothrombin. Several strategies can help preserve enzymatic function:

• Incorporate protease inhibitors (except serine protease inhibitors) throughout the purification process to prevent degradation

• Maintain a controlled temperature range (typically 4°C during purification steps)

• Include stabilizing agents such as glycerol (10-20%) or specific buffer additives

• Consider arginine or trehalose addition to prevent aggregation

• Optimize pH conditions to maintain structural integrity (typically pH 7.0-8.0)

• Employ gentle elution conditions during affinity chromatography to avoid denaturation

For long-term storage, researchers should validate the most appropriate method for their specific preparation:

• Flash freezing in small aliquots with 20% glycerol at -80°C

• Lyophilization with appropriate cryoprotectants

• Avoid repeated freeze-thaw cycles

Activity monitoring through fibrinogen-clotting assays at each stage of purification and storage provides critical feedback on protocol effectiveness. The target should be maintaining specific fibrinogen-clotting activity equivalent to 814-904 NIH alpha-thrombin units/mg .

What controls are essential when investigating bothrombin's effects in complex experimental systems?

When investigating bothrombin's effects in complex experimental systems, several essential controls should be implemented:

• Inhibitor controls:

  • Diisopropyl fluorophosphate treatment to confirm serine protease-dependent effects

  • Hirudin addition to differentiate bothrombin effects from potential alpha-thrombin contamination

• Substrate specificity controls:

  • Monitoring both fibrinopeptide A and B release to confirm selective cleavage pattern

  • Using fibrinogen variants with modified cleavage sites

• Platelet interaction controls:

  • Anti-GP IIb/IIIa and anti-GP Ib monoclonal antibodies to block specific interaction pathways

  • Prostaglandin E1 (1 μM) or EDTA (10 mM) treatments to verify calcium and activation dependence

  • Platelets from Bernard-Soulier syndrome patients as a negative control for GP Ib-dependent effects

• Recombinant protein-specific controls:

  • Empty vector expression product to control for host cell protein contamination

  • Tag-only constructs to verify tag effects

  • Heat-inactivated enzyme to control for non-enzymatic protein effects

• Dose-response analysis:

  • Establishing dose-dependent effects to confirm specific activity relationships

  • Determining minimal effective concentrations for different experimental endpoints

These controls help distinguish specific bothrombin-mediated effects from background processes and confirm the mechanisms underlying observed phenomena.

How can researchers reconcile contradictory data when comparing native and recombinant bothrombin?

Researchers may encounter contradictory data when comparing native bothrombin (purified from Bothrops jararaca venom) with recombinant bothrombin expressed in E. coli. Several methodological approaches can help reconcile these discrepancies:

• Glycosylation analysis: Native bothrombin contains three Asn-linked oligosaccharide chains , while E. coli-expressed protein lacks glycosylation . Comparative glycosylation analysis using lectins or mass spectrometry can quantify these differences.

• Structural characterization: Circular dichroism spectroscopy, limited proteolysis, and thermal stability assays can reveal differences in protein folding and stability between native and recombinant forms.

• Tag interference assessment: The N-terminal 10xHis-tag and C-terminal Myc-tag on recombinant bothrombin may affect certain activities. Creating tag-cleavable constructs allows direct comparison pre- and post-tag removal.

• Activity normalization: Rather than comparing equal protein amounts, normalizing based on specific activity (NIH alpha-thrombin units/mg) provides more reliable functional comparisons.

• Parallel characterization table:

By systematically addressing these potential sources of variation, researchers can develop a more nuanced understanding of structure-function relationships and reconcile apparently contradictory observations.