Recombinant Human Superoxide dismutase [Cu-Zn] (SOD1) (Active)

What is the molecular structure of recombinant human SOD1?

Recombinant Human SOD1 is a 16.8 kDa protein containing 160 amino acid residues when expressed with an N-terminal His-tag. The native protein consists of 154 amino acids and functions as a homodimer, with each monomer binding one copper and one zinc ion. The enzyme belongs to the Cu-Zn superoxide dismutase family and adopts a characteristic β-barrel structure with metal-binding sites crucial for its catalytic activity . The recombinant protein's primary sequence is identical to native human SOD1, though expression systems may introduce minor modifications such as affinity tags to facilitate purification.

How does recombinant SOD1 catalyze the dismutation of superoxide radicals?

Recombinant SOD1 catalyzes the conversion of superoxide radicals (O₂⁻) to molecular oxygen (O₂) and hydrogen peroxide (H₂O₂) through a two-step redox mechanism. In the first step, the enzyme's copper ion is reduced by a superoxide radical, generating oxygen. In the second step, another superoxide radical reacts with the reduced copper ion, along with two protons, to form hydrogen peroxide while regenerating the oxidized copper. This cyclic catalytic process efficiently neutralizes harmful superoxide radicals at a rate approaching diffusion-limited kinetics, making SOD1 one of the most efficient enzymes known . The zinc ion, while not directly involved in catalysis, plays a critical structural role in maintaining the active site geometry.

What are the critical considerations for maintaining the activity of recombinant SOD1 during purification?

Maintaining SOD1 activity during purification requires careful attention to several factors. First, copper and zinc ions must be present throughout the purification process to prevent metal loss, which would compromise enzymatic activity. Second, reducing agents must be used judiciously—while they prevent unwanted oxidation of cysteine residues, excessive exposure can disrupt the intramolecular disulfide bond essential for structural stability. Third, pH should be maintained between 7.0-8.0, as extreme pH conditions can cause metal loss or protein denaturation. Fourth, purification should be conducted at 4°C to minimize proteolytic degradation . Activity should be verified post-purification using assays such as the pyrogallol method, with active preparations demonstrating activity levels not less than 1.0 × 10⁴ IU/mg .

How do post-translational modifications affect recombinant SOD1 structure and function?

Post-translational modifications significantly impact SOD1 structure and function. Studies of human erythrocyte SOD1 reveal that the enzyme is phosphorylated at threonine 2 and potentially at either threonine 58 or serine 59, while also being glutathionylated at cysteine 111 . These modifications have profound effects: cysteine 111 glutathionylation increases the dissociation constant (Kd) of the SOD1 dimer by 2-fold, promoting monomer formation and potentially initiating the aggregation process implicated in ALS pathogenesis. This modification results in a 67% increase in monomer concentration at physiological conditions . Recombinant SOD1 expressed in E. coli lacks these modifications, which may limit its utility in certain experimental paradigms studying disease mechanisms. Researchers should carefully consider whether their experimental questions require a recombinant protein with native post-translational modifications.

What techniques can accurately identify and quantify post-translational modifications in recombinant SOD1?

A comprehensive approach to identifying post-translational modifications in recombinant SOD1 combines "bottom-up" and "top-down" mass spectrometry techniques. In the bottom-up approach, the protein is enzymatically digested into peptides that are analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS), allowing for precise localization of modifications. The top-down approach analyzes intact protein by high-resolution mass spectrometry to provide information about modification stoichiometry . To quantify specific modifications like glutathionylation, researchers can employ differential alkylation strategies followed by mass spectrometric analysis. For phosphorylation, a combination of titanium dioxide enrichment and multiple reaction monitoring (MRM) mass spectrometry provides sensitive quantification. Western blotting with modification-specific antibodies can complement mass spectrometric approaches for routine monitoring of key modifications .

How effective is recombinant SOD1 in reducing reperfusion injury in experimental models?

Recombinant human SOD1 demonstrates significant efficacy in reducing reperfusion injury across multiple experimental models. In isolated heart studies using electron paramagnetic resonance spectroscopy, administration of r-h-SOD during reperfusion reduced oxygen-centered alkyl peroxyl free radical concentrations by 49% (from 6.8±0.3 μM to 3.5±0.3 μM) and nitrogen-centered free radical concentrations by 38% (from 3.4±0.3 to 2.1±0.3 μM) . The protective effect requires the specific enzymatic activity of SOD1, as demonstrated by the lack of protection with peroxide-inactivated r-h-SOD. The greatest efficacy is observed when SOD1 is administered at the onset of reperfusion, coinciding with the burst of free radical generation that peaks at 10 seconds after reperfusion begins . Co-administration with catalase, which removes hydrogen peroxide generated by SOD1 activity, further enhances protection by preventing secondary reactive oxygen species formation .

What are the pharmacokinetic considerations when using recombinant SOD1 in animal models?

The pharmacokinetic profile of recombinant SOD1 presents significant challenges for therapeutic applications. Cu/ZnSOD has a remarkably short plasma half-life of only 6-10 minutes in rats, limiting its bioavailability . This rapid clearance necessitates continuous infusion or frequent dosing to maintain therapeutic levels. In contrast, the mitochondrial isoform MnSOD demonstrates a substantially longer half-life of 5-6 hours, making it potentially more suitable for treating chronic conditions . Various strategies have been developed to extend SOD1's half-life, including PEGylation, liposomal encapsulation, and fusion to cell-penetrating peptides or albumin-binding domains. When designing experiments, researchers should consider these pharmacokinetic limitations and implement appropriate delivery strategies to ensure sufficient enzyme activity reaches the target tissues. Monitoring SOD1 activity in plasma and target tissues throughout the experimental timeline is essential for accurate interpretation of results.

How do SOD1 mutations contribute to ALS pathogenesis, and what can we learn from recombinant mutant proteins?

SOD1 mutations contribute to ALS pathogenesis through multiple mechanisms that can be studied using recombinant mutant proteins. Over 100 different SOD1 mutations have been identified in familial ALS cases, with each mutation potentially affecting protein stability, metal binding, dimerization, or catalytic activity differently . Recombinant mutant SOD1 proteins allow researchers to systematically investigate these properties. Studies have revealed that many ALS-associated SOD1 mutations promote protein aggregation by destabilizing the dimer interface, increasing the population of monomeric species that are prone to misfolding . Interestingly, certain pathogenic SOD1 variants (Arg-38, Arg-47, Arg-86, and Ala-94) are specifically polyubiquitinated by RNF19A and MARCH5, targeting them for proteasomal degradation . This suggests cellular quality control mechanisms recognize these variants as aberrant. Recombinant SOD1 mutants serve as valuable tools for screening potential therapeutics and understanding the molecular basis of SOD1-mediated neurodegeneration.

What is the evidence for SOD1 involvement in sporadic ALS, and how can recombinant SOD1 be used to study this connection?

Recent evidence suggests SOD1 involvement in sporadic ALS (sALS), which accounts for 90% of all ALS cases, despite the absence of SOD1 mutations. Groundbreaking research using real-time quaking-induced conversion (RT-QuIC) assays has detected prion-like SOD1 aggregates in postmortem spinal cord and motor cortex tissues from sALS patients . This unexpected finding challenges the previous dogma that sALS is primarily associated with TDP-43 aggregation rather than SOD1 pathology. Additionally, studies have shown altered SOD1 mRNA expression in nervous tissues affected by ALS, with elevated levels in the brain stem and spinal cord of sALS patients . Recombinant SOD1 can be used to study this connection by serving as a substrate in seed amplification assays to detect misfolded SOD1 species in patient biosamples, potentially enabling earlier diagnosis. Furthermore, recombinant SOD1 subjected to oxidative stress in vitro can model the overoxidized forms hypothesized to trigger sporadic ALS , allowing investigation of aggregation mechanisms and therapeutic strategies relevant to both familial and sporadic forms of the disease.

How can researchers accurately distinguish between the effects of recombinant SOD1's enzymatic activity versus non-enzymatic properties in experimental systems?

Distinguishing between enzymatic and non-enzymatic effects of recombinant SOD1 requires careful experimental design incorporating appropriate controls. The gold standard approach utilizes enzymatically inactive SOD1 variants created either by peroxide-inactivation of the active enzyme or through site-directed mutagenesis of critical active site residues (H46A/H48A copper-binding mutants or H63A/H71A/H80A zinc-binding mutants). These inactive variants maintain the protein's structural properties while lacking catalytic activity. Alternative approaches include using SOD1 inhibitors like diethyldithiocarbamate (DDC) or competitive SOD mimetics like MnTBAP. To control for potential contaminants in recombinant preparations, researchers should compare multiple production batches and expression systems. When studying potential signaling roles of SOD1, receptor-binding domains can be blocked using specific antibodies or peptides without affecting enzymatic activity. Comparative dose-response studies between wild-type and inactive variants can further elucidate which effects correlate with enzymatic activity versus protein concentration, helping distinguish catalytic from structural or signaling roles.

How does recombinant SOD1 interact with other antioxidant enzymes in experimental systems?

Recombinant SOD1 functions within a complex antioxidant network, interacting with multiple enzymes in experimental systems. Its primary interaction is with catalase, which decomposes the hydrogen peroxide produced by SOD1's dismutation of superoxide. This partnership is so critical that SOD1 administration without adequate catalase can potentially increase oxidative damage due to H₂O₂ accumulation . Studies show that co-administration of SOD1 with catalase reduces free radical concentrations more effectively than SOD1 alone, reducing PBN spin adduct concentration to 18% of control values compared to 44% with bovine Cu-Zn superoxide dismutase plus catalase . SOD1 also interacts functionally with glutathione peroxidase, which provides an alternative pathway for H₂O₂ elimination, particularly in cells with lower catalase activity. Furthermore, thioredoxin reductase systems interact with SOD1 by maintaining its redox state. When designing antioxidant interventions, researchers should consider the relative expression and activity levels of these complementary enzymes in their experimental system, as the protective effect of recombinant SOD1 depends on the capacity of downstream enzymes to process H₂O₂.

What are the mechanisms by which oxidative stress can modify SOD1 structure and function, and how can these modifications be studied using recombinant protein?

Oxidative stress can modify SOD1 through several mechanisms, each potentially contributing to altered function and aggregation propensity. These modifications include oxidation of metal-coordinating histidine residues leading to metal loss, oxidation of free cysteines (particularly Cys111) promoting disulfide cross-linking between monomers, glutathionylation of Cys111 destabilizing the dimer interface, and oxidation of tryptophan and tyrosine residues affecting protein folding . Recent research has demonstrated that hypochlorous acid (HOCl) treatment of wild-type SOD1 facilitates filament formation, suggesting that overoxidized SOD1 may be a triggering factor in sporadic ALS . These modifications can be studied using recombinant SOD1 subjected to controlled oxidative conditions in vitro, followed by mass spectrometric analysis to identify specific oxidation sites. Structural changes can be monitored using circular dichroism, fluorescence spectroscopy, and differential scanning calorimetry. Aggregation propensity can be assessed through thioflavin T binding assays, dynamic light scattering, and electron microscopy. By systematically introducing specific oxidative modifications to recombinant SOD1, researchers can dissect their individual contributions to protein dysfunction and establish structure-function relationships relevant to oxidative stress-related diseases.

How do metal ions influence the structural stability and aggregation propensity of recombinant SOD1?

Metal ions play a crucial role in SOD1 structural stability and aggregation propensity through complex mechanisms that extend beyond catalytic function. Each SOD1 monomer binds one copper and one zinc ion, with copper coordinated by histidines 46, 48, 63, and 120, while zinc binds to histidines 63, 71, 80, and aspartate 83 . Metal-depleted (apo) SOD1 shows dramatically reduced thermal stability, with melting temperatures decreased by up to 20°C compared to fully metalated enzyme. Zinc binding primarily enhances structural stability, while copper is essential for catalytic activity. The absence of zinc significantly increases the population of partially unfolded intermediates prone to aggregation . Metal loss also disrupts the intramolecular disulfide bond between Cys57 and Cys146, further destabilizing the protein. In ALS-associated mutants, these effects are often exacerbated, creating a mechanistic link between metal homeostasis and disease. Recombinant SOD1 can be prepared with different metalation states (Cu,Zn-SOD1, E,Zn-SOD1, Cu,E-SOD1, or E,E-SOD1, where E represents an empty binding site) by controlling metal availability during expression and purification or through chelation and reconstitution approaches. This allows systematic investigation of how specific metal ions influence stability, activity, and aggregation under various experimental conditions relevant to neurodegenerative disease research.

What strategies are most effective for enhancing the tissue penetration and half-life of recombinant SOD1 for therapeutic applications?

Several strategies have proven effective for enhancing the tissue penetration and half-life of recombinant SOD1. Polyethylene glycol (PEG) conjugation significantly extends SOD1's plasma half-life from minutes to hours by increasing molecular size and shielding the protein from proteolytic degradation and immune recognition . Liposomal encapsulation not only protects SOD1 from degradation but also facilitates cellular uptake and can be targeted to specific tissues through surface modifications. Fusion of SOD1 with cell-penetrating peptides (CPPs) such as TAT or polyarginine sequences enhances cellular uptake and potentially allows SOD1 to reach intracellular compartments where free radicals are generated. Recombinant fusion proteins combining SOD1 with albumin-binding domains exploit albumin's natural long circulation time. For neurological applications, fusion with antibody fragments targeting transferrin or insulin receptors facilitates blood-brain barrier crossing. Comparative studies in animal models of ischemia-reperfusion injury have demonstrated that PEGylated SOD1 provides superior tissue protection compared to native enzyme, correlating with its extended half-life and improved tissue distribution . The choice of delivery strategy should be guided by the specific therapeutic application, target tissue, and required duration of action.

How can researchers address the challenges of immunogenicity when using recombinant human SOD1 in animal models?

Immunogenicity presents a significant challenge when using recombinant human SOD1 in animal models, particularly for long-term studies. Researchers can employ several strategies to address this issue. First, using species-matched SOD1 (e.g., mouse SOD1 in mouse models) eliminates immunogenicity but may not accurately represent human therapeutic applications. Second, immunosuppressive agents can be co-administered, though this approach may interfere with the experimental disease model, particularly in inflammation-related studies. Third, PEGylation not only extends half-life but also masks immunogenic epitopes, reducing antibody generation . Fourth, incorporating SOD1 into stealth liposomes or nanoparticles shields the protein from immune recognition while maintaining activity. Fifth, site-specific mutations of known immunogenic epitopes that don't affect enzymatic activity can reduce immunogenicity. To monitor immunogenicity during experiments, researchers should regularly collect serum samples for anti-SOD1 antibody detection using ELISA or Western blotting. A biphasic loss of therapeutic effect often indicates antibody development, necessitating increased dosing or alternative delivery strategies. For translational studies, humanized animal models expressing human immune components provide more predictive immunogenicity assessment.

How might recombinant SOD1 contribute to the development of diagnostic biomarkers for neurodegenerative diseases?

Recombinant SOD1 is playing a pivotal role in developing diagnostic biomarkers for neurodegenerative diseases, particularly ALS. The most promising approach utilizes recombinant SOD1 as a substrate in seed amplification assays such as real-time quaking-induced conversion (RT-QuIC), which can detect vanishingly small amounts of misfolded SOD1 "seeds" in patient biosamples . This technique has recently demonstrated that SOD1 aggregates are present not only in patients with SOD1 mutations but also in sporadic ALS and C9orf72-linked familial ALS patients, suggesting broader SOD1 involvement in ALS pathology than previously recognized . Recombinant SOD1 can also be used to develop and standardize antibody-based assays targeting misfolded SOD1 conformers in cerebrospinal fluid, with several conformation-specific antibodies already showing promise in distinguishing ALS patients from controls. Furthermore, recombinant SOD1 serves as a crucial positive control for establishing assay sensitivity and specificity in measuring SOD1 post-translational modifications that may serve as disease biomarkers . By enabling early detection of pathological SOD1 species before symptom onset, these biomarker approaches could revolutionize clinical trials by identifying candidates for preventive therapies and allowing intervention before irreversible neuronal loss occurs.

What novel applications of recombinant SOD1 are emerging in nanomedicine and biomaterial science?

Recombinant SOD1 is finding innovative applications at the intersection of nanomedicine and biomaterial science. SOD1-conjugated nanoparticles represent a cutting-edge approach for targeted antioxidant delivery, with recombinant SOD1 being attached to gold, silica, or polymeric nanoparticles through various coupling chemistries to maintain enzymatic activity while improving stability and cellular uptake. SOD1-functionalized biomaterials are being developed for tissue engineering applications, where controlling oxidative stress is crucial for cell survival and differentiation. For example, SOD1-incorporating hydrogels show enhanced support of neural cell growth under oxidative stress conditions relevant to spinal cord injury repair. In implantable biosensors, SOD1 coatings reduce the foreign body response by neutralizing inflammatory cell-derived superoxide, extending sensor lifespan and accuracy. SOD1-based nanozymes, created by incorporating the enzyme into metal-organic frameworks or mesoporous silica, demonstrate enhanced stability under harsh conditions while maintaining catalytic activity. Perhaps most intriguingly, stimulus-responsive SOD1 delivery systems activated by specific pathological conditions (such as acidic pH or elevated reactive oxygen species) are being engineered to provide on-demand antioxidant activity only when and where needed, maximizing therapeutic efficacy while minimizing potential side effects from systemic antioxidant administration .