SOD Human His

What is SOD1 and what is its function in human cells?

Human Cu/Zn Superoxide Dismutase (SOD1) is an essential enzyme that catalyzes the reaction between superoxide anions and hydrogen to yield molecular oxygen and hydrogen peroxide . This 189-amino acid protein functions primarily as a cellular defense mechanism, protecting cells against dangerous levels of superoxide radicals . SOD1 binds copper and zinc ions and is one of three isozymes responsible for neutralizing supercharged oxygen molecules in the body, which would otherwise cause cellular damage if their levels remained uncontrolled .

The methodological approach to studying SOD1 function typically involves:

• Enzyme activity assays using spectrophotometric methods

• Cellular oxidative stress models

• Genetic manipulation in model organisms

• Structural analysis using X-ray crystallography and NMR

What is the significance of the His-tag in recombinant human SOD1?

The His-tag in recombinant human SOD1 consists of 10 histidine residues added to the N-terminus of the protein, resulting in a molecular mass of approximately 40.0kDa . This modification serves several critical research purposes:

• Purification efficiency: The His-tag allows for single-step purification using affinity chromatography techniques

• Detection capability: It enables antibody-based detection methods with high specificity

• Structural studies: Facilitates protein immobilization for structural and interaction analyses

• Protein-protein interaction studies: Can be used in pull-down assays to identify binding partners

When working with His-tagged SOD1, researchers should consider that while the tag facilitates purification, it may potentially affect certain protein properties compared to the native form.

How should researchers properly reconstitute and store SOD Human His?

Proper handling of SOD Human His is crucial for maintaining its biological activity. The lyophilized protein, while stable at room temperature for up to 3 weeks, should be stored desiccated below -18°C for long-term stability . For reconstitution, researchers should:

• Reconstitute the lyophilized SOD Human His in sterile 18MΩ-cm H₂O at a concentration of at least 100μg/ml

• Further dilute this stock in appropriate aqueous solutions for experimental use

• Store reconstituted protein at 4°C if using within 2-7 days

• For longer storage, maintain at temperatures below -18°C

• Critically, prevent freeze-thaw cycles which significantly reduce protein activity

This methodology ensures optimal enzyme activity and experimental reproducibility when working with SOD Human His preparations.

What experimental models are commonly used to study SOD1 function?

Several experimental models have proven valuable for studying SOD1 function, with the SOD-deficient yeast system being particularly informative:

• S. cerevisiae SOD1-deficient models: The sod1Δ mutant strain (EG118) derived from wild-type EG103 exhibits specific amino acid auxotrophies (lysine and methionine) that can be complemented by functional SOD or SOD mimics . This provides a clear phenotypic readout for SOD activity.

• Methodological approach for yeast models:

  • Culture cells in 96-well plates containing SD medium supplemented with all amino acids except methionine

  • Maintain aerobic growth conditions at 30°C with shaking at 220 rpm

  • Periodically disrupt cell clumping using a specialized 96-pin stirrer

  • Monitor growth turbidimetrically at 600 nm using an ELISA reader

• Mammalian cell culture models: Including neuronal cell lines and primary cultures

• In vivo models: SOD1 transgenic mice and other animal models of ALS

The choice of model depends on the specific research question, with yeast models offering high-throughput screening capability and mammalian models providing greater physiological relevance.

What methodological approaches are most effective for studying SOD1 in neurodegenerative diseases?

SOD1 mutations are directly linked to familial amyotrophic lateral sclerosis (ALS), making methodological approaches to studying SOD1 in neurodegeneration critically important . Effective research strategies include:

• Clinical biomarker monitoring: Recent clinical trials for SOD1-ALS, such as the ALN-SOD study, monitor both SOD1 protein levels and neurofilament light chain levels as markers of disease progression and treatment response .

• Genetic therapeutic approaches: Intrathecal administration of gene-targeting compounds (like ALN-SOD) requires specialized methodological considerations for delivery and assessment .

• Combined biochemical and clinical assessments: The most robust approaches integrate:

  • Protein aggregation analysis

  • Oxidative stress biomarkers

  • Neurological function assessments

  • Longitudinal monitoring of disease progression

• Multi-omics approaches: Integration of proteomics, transcriptomics, and metabolomics data to understand disease mechanisms

Clinical research with SOD1-ALS patients typically requires a placebo-controlled design with regular monitoring and precise measurement of protein biomarkers to evaluate therapeutic efficacy .

How can SOD mimics be effectively evaluated for therapeutic potential?

The evaluation of SOD mimics represents an important avenue for developing therapeutics for conditions involving oxidative stress. A systematic methodological approach includes:

• In vitro enzymatic activity: Assessment of superoxide dismutation rates using standard biochemical assays.

• Yeast complementation assays: Using sod1Δ S. cerevisiae strains to test whether compounds can restore growth in media lacking methionine or lysine . This biologically relevant screening method specifically measures the compound's ability to functionally replace SOD:

  • Compounds must catalyze superoxide dismutation at rates higher than spontaneous dismutation

  • Only truly effective mimics will restore aerobic growth in auxotrophic media

  • Growth restoration directly correlates with therapeutic potential

• Quantitative structure-activity relationship (QSAR) studies: Correlation between structural features and SOD-like activity.

• Combined approaches: The most robust methodological approach integrates both in vitro biochemical assays and biological models to establish that "the SOD-like activity parallels therapeutic potential" .

This methodological framework allows researchers to systematically evaluate candidate compounds beyond simple in vitro activity measurements.

What challenges exist in using recombinant SOD1 with His-tag for structural studies?

Structural studies of His-tagged SOD1 present several methodological challenges that researchers must address:

• Tag interference: The 10×His tag (21 additional amino acids) at the N-terminus can potentially alter structural properties compared to native SOD1 . Researchers should consider:

  • Conducting parallel studies with tagged and untagged proteins

  • Using tag-cleavage methods when appropriate

  • Verifying that tag position doesn't interfere with metal binding sites

• Protein purity considerations: While His-tagged SOD1 can achieve >95% purity as determined by SDS-PAGE and HPLC analyses , residual contaminants may affect structural studies, necessitating additional purification steps.

• Metal binding stoichiometry: Ensuring proper copper and zinc incorporation remains challenging but is essential for structural and functional relevance.

• Aggregation tendencies: SOD1, particularly mutant forms, can form aggregates that complicate structural analyses. Researchers should implement:

  • Dynamic light scattering to monitor aggregation state

  • Size-exclusion chromatography to separate monomeric protein

  • Optimized buffer conditions to minimize aggregation

What are the latest approaches in clinical trials for SOD1-related ALS?

Recent clinical trials for SOD1-related ALS demonstrate sophisticated methodological approaches that can inform broader research efforts:

• Target-specific therapeutics: The ALN-SOD clinical trial specifically targets people with SOD1 gene mutations (SOD1-ALS) . This precision medicine approach requires:

  • Genetic confirmation of SOD1 mutations

  • Stratification of participants based on mutation type

  • Tailored outcome measures relevant to SOD1-ALS progression

• Administration methodology: Intrathecal delivery (injection into the space around the spinal cord) provides direct access to the central nervous system, bypassing the blood-brain barrier . This specialized delivery method requires:

  • Standardized protocols for administration

  • Safety monitoring specific to the delivery route

  • Pharmacokinetic analysis in cerebrospinal fluid

• Biomarker evaluation: Multiple-dose studies assess SOD1 protein levels and neurofilament light chain as quantitative measures of treatment effect , allowing for:

  • Dose-response relationship analysis

  • Target engagement confirmation

  • Potential surrogate endpoint development

• Safety and efficacy assessment: Comprehensive evaluations through regular check-ups and specialized testing to monitor both adverse effects and therapeutic benefits .

These methodological approaches represent the current standard for clinical investigation of SOD1-targeted therapeutics and can inform preclinical research design.

Experimental Models for SOD1 Research

SOD1 Protein Stability and Storage Conditions

How can researchers address protein activity loss in SOD Human His preparations?

SOD Human His activity can be compromised by several factors, requiring systematic troubleshooting approaches:

• Freeze-thaw cycles: Each cycle significantly reduces enzyme activity . Implement:

  • Single-use aliquots of reconstituted protein

  • Rapid thawing techniques without excessive warming

  • Addition of cryoprotectants for freezing stocks

• Metal dissociation: Copper and zinc are essential for SOD1 activity:

  • Consider supplementing buffers with appropriate metal ions

  • Use metal chelators (EDTA, EGTA) cautiously

  • Monitor metal content using spectroscopic methods

• Oxidative damage to the enzyme itself: Paradoxically, SOD1 can be damaged by the species it neutralizes:

  • Include reducing agents in buffers when appropriate

  • Minimize exposure to strong oxidants during purification

  • Prepare fresh solutions for critical experiments

• Concentration-dependent effects: Maintain protein above critical concentration (100μg/ml recommended) to preserve stability .

What methodological considerations apply when comparing SOD mimics to recombinant SOD1?

When evaluating SOD mimics against recombinant SOD Human His, several methodological considerations are essential:

• Activity normalization: Calculate and compare:

  • Catalytic rate constants (kcat)

  • Catalytic efficiency (kcat/Km)

  • Activity per molecular weight

  • Activity under physiological conditions

• Biological relevance testing: The yeast complementation assay provides a functional biological readout:

  • Only compounds catalyzing dismutation faster than spontaneous rates will show activity

  • Growth restoration in auxotrophic media directly correlates with mimetic effectiveness

  • Controls should include wild-type yeast (EG103) and untreated sod1Δ mutants (EG118)

• Delivery and bioavailability: Consider:

  • Membrane permeability differences

  • Cellular distribution patterns

  • Stability in biological media

  • Potential off-target effects

• Combined methodological approach: Integrate in vitro biochemical assays with biological models to establish that "SOD-like activity parallels therapeutic potential" .