SNAP-8

Research Overview

SNAP-8 serves as a valuable research tool for investigating neurotransmitter release mechanisms and neuromuscular signaling in laboratory settings. This synthetic octapeptide represents an elongated, optimized analog of acetyl hexapeptide-3 (Argireline), designed to provide enhanced activity in SNARE complex modulation studies. Research applications span neuromuscular function investigation, synaptic vesicle fusion mechanism studies, acetylcholine release dynamics, and cosmetic peptide research examining muscle contraction-dependent expression line formation.

The peptide’s designation relates to its mechanism of action: SNAP-8 mimics the N-terminal domain of SNAP-25 (Synaptosome-Associated Protein 25kDa), one of three core SNARE (Soluble N-ethylmaleimide-sensitive factor Attachment protein REceptor) proteins essential for synaptic vesicle membrane fusion. By acting as a competitive substrate, SNAP-8 interferes with SNARE complex formation, modulating neurotransmitter release efficiency at neuronal synapses and neuromuscular junctions. Laboratory studies investigate these effects on acetylcholine release, muscle fiber contraction, and downstream cellular signaling pathways.

Research applications have expanded from initial cosmetic peptide investigations to broader neuromuscular research. Studies examine SNAP-8’s effects on neurotransmitter vesicle docking, membrane fusion mechanisms, calcium-dependent exocytosis, and muscle contraction signaling. Research protocols employ in vitro neuromuscular preparations, electrophysiological recordings, cell culture systems, and appropriate preclinical models to characterize biological activities and mechanism of action.

Molecular Characteristics

Complete Specifications:

• CAS Registry Number: 868844-74-0
• Molecular Weight: 1,075.2 Da
• Molecular Formula: C47H75N15O14
• Amino Acid Sequence: Ac-Glu-Glu-Met-Gln-Arg-Arg-Ala-Asp-NH2
• Alternative Names: Acetyl octapeptide-3, SNAP-8 peptide
• Peptide Classification: Synthetic octapeptide, SNARE complex modulator
• Appearance: White to off-white lyophilized powder
• Solubility: Water, bacteriostatic water, phosphate buffered saline
• Net Charge: Negative at physiological pH (contains acidic residues)

The peptide’s 8-amino acid structure incorporates key sequence elements critical for SNARE protein interaction. The N-terminal acetylation (Ac-) and C-terminal amidation (-NH2) modifications enhance stability and receptor interaction specificity compared to unmodified peptide sequences. Two consecutive glutamic acid residues (positions 1-2) provide acidic character and charge distribution important for SNARE protein recognition. The methionine residue (position 3) represents a potentially oxidation-sensitive site requiring careful handling. Two arginine residues (positions 5-6) contribute positive charge and participate in electrostatic interactions with SNARE complex components.

The sequence design specifically targets the SNAP-25 binding interface within the SNARE complex. SNAP-25 contributes two alpha-helical domains to the four-helix bundle structure of the synaptic SNARE complex, alongside single helices from syntaxin-1 and synaptobrevin-2 (VAMP-2). SNAP-8’s mimicry of the N-terminal SNAP-25 region allows it to compete for binding sites, interfering with complete SNARE complex assembly and subsequent membrane fusion.

SNARE Complex Mechanism and Research Significance

SNARE Complex Function:

The SNARE complex represents one of the most extensively studied protein complexes in neurobiology, serving as the minimal membrane fusion machinery for neurotransmitter release. Understanding SNARE function is fundamental to neurotransmitter release research:

• Complex Components: The neuronal SNARE complex consists of SNAP-25, syntaxin-1 (both on the plasma membrane, termed t-SNAREs for target membrane), and synaptobrevin-2/VAMP-2 (on synaptic vesicles, termed v-SNARE for vesicle membrane).

• Fusion Mechanism: These three proteins assemble into a tight four-helix bundle that brings vesicle and plasma membranes into close proximity. The energy released during SNARE complex formation provides the mechanical force to overcome the energy barrier for membrane fusion.

• Calcium Regulation: While SNARE complex formation primes vesicles for release, calcium influx through voltage-gated calcium channels triggers the final fusion event through calcium-sensing proteins like synaptotagmin.

SNAP-8 Research Applications in SNARE Studies:

SNAP-8 provides a tool for investigating SNARE complex function through competitive inhibition:

• Complex Formation Studies: Research examining how SNAP-8 interferes with endogenous SNARE protein assembly, providing insights into binding determinants and assembly kinetics.

• Dose-Response Characterization: Studies establishing concentration-dependent effects on neurotransmitter release, generating IC50 values for SNARE complex inhibition.

• Temporal Dynamics: Investigation of SNAP-8’s effects on rapid vs. slow synaptic vesicle release pools, distinguishing between readily releasable and reserve vesicle populations.

• Specificity Studies: Research determining selectivity for neuronal SNARE complexes vs. other SNARE variants involved in different trafficking pathways.

Research Applications

Neuromuscular Function and Synaptic Transmission Research

SNAP-8 serves as a research tool for investigating neuromuscular signaling and acetylcholine release mechanisms:

• Neurotransmitter Release Studies: Investigation of acetylcholine release dynamics at neuromuscular junctions using electrophysiological recordings (endplate potentials, miniature endplate potentials).

• Muscle Contraction Research: Analysis of muscle fiber contraction force, contraction kinetics, and calcium signaling following neurotransmitter release modulation.

• Synaptic Vesicle Dynamics: Studies examining vesicle docking, priming, fusion, and recycling processes using fluorescent imaging techniques and electron microscopy.

• Calcium-Dependent Exocytosis: Research investigating the relationship between calcium influx, synaptotagmin activation, and SNARE-mediated membrane fusion.

• Neuromuscular Junction Morphology: Examination of structural changes at motor endplates, including acetylcholine receptor clustering and synaptic cleft dimensions.

Research protocols typically employ isolated neuromuscular preparations (frog sartorius muscle, mouse diaphragm), patch-clamp electrophysiology, myography for force measurements, and high-resolution imaging techniques to characterize SNAP-8’s effects on neurotransmitter release and muscle contraction.

Cosmetic Peptide Research Applications

SNAP-8 research extends to cosmetic peptide investigations examining expression line formation:

• Expression Line Mechanism Studies: Investigation of how repeated facial muscle contractions contribute to expression line formation (forehead lines, crow’s feet, frown lines).

• Dermal-Epidermal Junction Research: Studies examining how mechanical forces from underlying muscle contractions affect dermal structure and collagen organization.

• Fibroblast Mechanotransduction: Research on how dermal fibroblasts respond to mechanical stress from muscle contractions, influencing collagen synthesis and degradation patterns.

• Topical Delivery Studies: Investigation of peptide penetration through stratum corneum, delivery optimization strategies, and bioavailability enhancement techniques.

• Efficacy Assessment Methods: Development of objective measurement techniques for expression line depth, including profilometry, high-resolution imaging, and 3D facial scanning.

Laboratory studies employ ex vivo skin penetration models, dermal fibroblast cell cultures subjected to mechanical stress, and appropriate preclinical models for efficacy and mechanism investigation.

Neurological Research Applications

Research applications encompass broader neurological investigations:

• Synaptic Plasticity Studies: Examination of how neurotransmitter release modulation affects synaptic strength, long-term potentiation (LTP), and long-term depression (LTD).

• Neurotransmitter Dynamics Research: Investigation of acetylcholine, glutamate, GABA, and other neurotransmitter release kinetics in various neuronal populations.

• Neuronal Circuit Function: Studies examining how synaptic transmission modulation affects circuit-level activity patterns and network synchronization.

• Disease Model Research: Investigation of SNARE complex dysfunction in neurological disease models where synaptic vesicle fusion is compromised.

• Botulinum Toxin Mechanism Comparison: Comparative research examining SNAP-8’s competitive inhibition mechanism vs. botulinum toxin’s proteolytic cleavage of SNARE proteins.

Research protocols utilize cultured neurons, brain slice electrophysiology, optogenetic stimulation combined with SNAP-8 treatment, and imaging techniques including fluorescent reporters of synaptic vesicle cycling.

Cell Biology and Vesicular Transport Research

SNAP-8 research contributes to fundamental cell biology investigations:

• Vesicle Fusion Mechanisms: Studies examining general principles of membrane fusion applicable beyond neurotransmitter release, including endosomal fusion, autophagy, and secretory vesicle fusion.

• SNARE Protein Interaction Studies: Research investigating protein-protein interaction determinants, binding interfaces, and conformational changes during complex assembly.

• Exocytosis Pathway Research: Examination of regulated vs. constitutive secretion pathways, comparing SNARE complex requirements across different cell types.

• Membrane Trafficking Studies: Investigation of vesicular transport mechanisms, cargo sorting, and fusion specificity in intracellular trafficking.

Laboratory approaches include biochemical binding assays, fluorescence resonance energy transfer (FRET) studies of SNARE complex assembly, liposome fusion assays, and live-cell imaging of vesicle dynamics.

Pharmacological Profile in Research Models

In Vitro Activity Characterization:

• Concentration Range: Research studies typically investigate SNAP-8 concentrations from nanomolar to micromolar ranges, depending on experimental system and endpoint measurements.

• Onset of Action: Effects on neurotransmitter release observable within minutes following application to neuromuscular preparations, reflecting time required for peptide diffusion and SNARE complex interference.

• Reversibility: Competitive inhibition mechanism suggests reversibility upon washout, though experimental verification depends on specific research models and tissue/cell preparations.

• Selectivity Profile: Research investigating selectivity for neuronal SNARE complexes vs. other SNARE-mediated fusion events in non-neuronal cells.

Structure-Activity Relationship Research:

Comparative studies with related peptides inform understanding of critical structural determinants:

• Acetyl Hexapeptide-3 (Argireline): Six-amino acid predecessor with similar mechanism but potentially different potency and duration profiles.

• Sequence Truncation Studies: Investigation of minimum peptide length required for SNARE complex inhibition.

• Amino Acid Substitution Research: Studies examining how specific amino acid changes affect binding affinity and inhibitory potency.

• Modification Effects: Research on how N-terminal acetylation and C-terminal amidation influence stability and activity compared to unmodified sequences.

Laboratory Handling and Storage Protocols

Lyophilized Powder Storage:

• Store at -20°C to -80°C in original sealed vial
• Protect from light exposure and moisture
• Desiccated storage environment required
• Stability data documented for 12+ months at -20°C
• Avoid repeated temperature cycling

Reconstitution Guidelines:

• Reconstitute with sterile water, bacteriostatic water (0.9% benzyl alcohol), or appropriate buffer (PBS pH 7.4)
• Add solvent slowly down vial side to minimize foaming and peptide adhesion to walls
• Gentle swirling motion recommended (avoid vigorous shaking or vortexing)
• Allow complete dissolution before use (typically 2-5 minutes)
• Final pH should be 7.0-8.0 for optimal stability
• Final concentration determination should account for peptide content assay results (typically 80-90%)

Reconstituted Solution Storage:

• Short-term storage: 4°C for up to 7 days (bacteriostatic water extends stability)
• Long-term storage: -20°C or -80°C in single-use aliquots
• Avoid repeated freeze-thaw cycles (maximum 2-3 cycles before significant activity loss)
• Consider addition of carrier proteins (BSA 0.1%) for dilute working solutions to minimize surface adsorption losses

Special Handling Considerations:

The methionine residue at position 3 represents a potential oxidation-sensitive site. Research protocols should consider:

• Minimize exposure to oxidizing conditions during storage and handling
• Consider addition of antioxidants (DTT, β-mercaptoethanol) for specific applications, though these may interfere with certain assays
• Monitor peptide quality through analytical methods if solutions are stored for extended periods
• Fresh reconstitution recommended for critical experiments requiring maximum activity

Quality Assurance and Analytical Testing

Each SNAP-8 batch undergoes comprehensive analytical characterization to ensure research-grade quality:

Purity Analysis:

• High-Performance Liquid Chromatography (HPLC): ≥98% purity
• Analytical method: Reversed-phase HPLC with UV detection at 220nm
• Chromatographic conditions: C18 column, acetonitrile/water gradient with 0.1% TFA
• Multiple peak integration ensuring accurate purity determination
• Related substance analysis identifying potential degradation products or synthesis impurities

Structural Verification:

• Electrospray Ionization Mass Spectrometry (ESI-MS): Confirms molecular weight 1,075.2 Da
• High-resolution mass spectrometry providing exact mass determination
• MS/MS fragmentation analysis for sequence verification
• Amino acid analysis: Quantitative determination of amino acid composition
• Peptide content determination: Quantifies actual peptide content by weight (typically 80-90% of total mass)

Contaminant Testing:

• Bacterial endotoxin: <5 EU/mg (LAL method, USP )
• Heavy metals: Below detection limits per USP or ICP-MS analysis
• Residual solvents: TFA, acetonitrile, and other synthesis solvents within ICH Q3C limits
• Water content: Karl Fischer titration (<8%, typically 3-6%)
• Bioburden testing for non-sterile research peptides

Documentation:

• Certificate of Analysis (COA) provided with each batch
• Lot-specific analytical data including chromatograms and mass spectra
• Third-party analytical verification available upon request for independent quality confirmation
• Stability data documented for recommended storage conditions
• Batch-specific QC results traceable by lot number for research record keeping

Research Considerations

Experimental Design Factors:

Researchers should carefully consider multiple factors when designing SNAP-8 experiments:

1. Concentration Selection: Determine appropriate concentration ranges based on research objectives, experimental system, and published literature. Consider pilot dose-response studies to establish optimal working concentrations for specific applications.

2. Exposure Timing: Design temporal protocols considering peptide diffusion time into tissues, binding kinetics to target sites, and duration of action in specific experimental systems.

3. Delivery Considerations: For in vivo or ex vivo tissue research, consider diffusion barriers, peptide stability in biological fluids, and potential enzymatic degradation requiring protease inhibitors.

4. Control Groups: Include comprehensive controls: vehicle-only (same buffer/solvent), positive controls (established modulators of neurotransmitter release), and scrambled peptide sequences when feasible.

5. Endpoint Selection: Choose appropriate outcome measures based on research questions: electrophysiological recordings (EPP/MEPP), muscle force measurements, calcium imaging, vesicle fusion assays, or morphological assessments.

Mechanism Investigation Approaches:

Understanding SNAP-8’s mechanism requires multi-faceted research approaches:

• Binding Studies: Investigation of SNAP-8 interaction with SNARE proteins using surface plasmon resonance (SPR), isothermal titration calorimetry (ITC), or co-immunoprecipitation approaches.

• SNARE Complex Assembly Assays: Biochemical assessment of ternary complex formation using purified SNARE proteins, examining how SNAP-8 interferes with complex assembly kinetics.

• Electrophysiology: Detailed characterization of effects on evoked and spontaneous neurotransmitter release using patch-clamp or sharp electrode recordings.

• Calcium Imaging: Studies examining whether SNAP-8 affects calcium influx or acts downstream at the fusion machinery level.

• Vesicle Tracking: Fluorescent imaging approaches (FM dyes, synaptic vesicle proteins tagged with fluorescent proteins) to visualize effects on vesicle mobilization and fusion.

Comparative Peptide Research:

Research comparing SNAP-8 with related compounds provides mechanistic insights:

• Acetyl Hexapeptide-3 Comparison: Studies distinguishing the eight-amino acid SNAP-8 from the six-amino acid predecessor in potency, duration, and specificity.

• Botulinum Toxin Mechanism Contrast: Comparative research examining reversible competitive inhibition (SNAP-8) vs. irreversible proteolytic cleavage (botulinum toxin) of SNARE proteins.

• Other Neuromuscular Modulators: Studies with acetylcholine receptor antagonists, calcium channel blockers, or other neurotransmitter release modulators to distinguish mechanisms and sites of action.

Compliance and Safety Information

Regulatory Status:
SNAP-8 is provided as a research chemical for in-vitro laboratory studies and preclinical research only. This product has not been approved by the FDA for human therapeutic use, cosmetic applications as an active ingredient requiring approval, dietary supplementation, or medical applications. Cosmetic peptide research applications must comply with appropriate regulatory frameworks.

Intended Use:

• In-vitro cell culture studies and biochemical assays
• Ex-vivo tissue preparations (isolated neuromuscular preparations, skin penetration studies)
• In-vivo preclinical research in approved animal models with appropriate IACUC protocols
• Laboratory investigation of neuromuscular mechanisms and SNARE complex function
• Cosmetic peptide mechanism research and efficacy studies
• Academic and institutional research applications

NOT Intended For:

• Human consumption or administration
• Cosmetic product formulation without appropriate regulatory compliance and safety testing
• Therapeutic treatment or diagnosis
• Dietary supplementation
• Veterinary therapeutic applications without appropriate oversight
• Any medical applications

Safety Protocols:
Researchers should follow standard laboratory safety practices when handling SNAP-8:

• Use appropriate personal protective equipment (lab coat, nitrile gloves, safety glasses)
• Handle in well-ventilated areas or fume hood when working with powders
• Follow institutional biosafety guidelines and chemical hygiene plans
• Dispose of waste according to local regulations for biological/chemical waste
• Consult material safety data sheet (MSDS) for additional safety information
• No significant acute toxicity anticipated, but handle as any research peptide with appropriate precautions

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