NAD+ Molecular Weight, Sequence & CAS Reference

Technical Summary & Core Molecular Specifications

Nicotinamide Adenine Dinucleotide (oxidized form, designated as NAD+) is a critical coenzyme found in all living cells, serving as a primary electron acceptor in cellular redox reactions and a required substrate for NAD+-dependent enzymes. When sourcing highly purified reagents for biochemical assays, analytical laboratories require definitive physical-chemical constants to calculate exact molarities and ensure experimental reproducibility across culture models.

The primary molecular specifications for research-grade NAD+ free acid are defined below:

• Chemical Formula: C21H27N7O14P2 • Exact Monoisotopic Mass: 663.1091 g/mol • Molecular Weight (Average): 663.43 g/mol • IUPAC Name: [[(2R,3S,4R,5R)-5-(6-aminopurin-9-yl)-3,4-dihydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl] [(2R,3S,4R,5R)-5-(3-carbamoylpyridin-1-ium-1-yl)-3,4-dihydroxyoxolan-2-yl]methyl hydrogen phosphate • CAS Registry Number: 53-84-9 (Free Acid); 20111-18-6 (Sodium Salt, Na-NAD+) • Structural Class: Pyridine-adenine dinucleotide coenzyme • Physical Form: Lyophilized white to off-white powder

Researchers evaluating structural parameters or ordering reference-grade materials can review our high-purity NAD+ product reference alongside our complete catalog of research peptides to support quantitative cellular assays.

Biochemical Structure: Amino Acid Sequence vs. Dinucleotide Architecture

A common point of inquiry among laboratory researchers navigating peptide and coenzyme libraries concerns the 'amino acid sequence' of NAD+. Strictly speaking, NAD+ is not a peptide and does not possess an amino acid sequence. It is a dinucleotide composed of two ribosyl rings linked through their 5' phosphate groups, with one ring attached to an adenine base and the other attached to a nicotinamide ring.

Because NAD+ lacks peptide bonds (amide linkages between alpha-amino acids), standard primary sequence notation (e.g., N-terminus to C-terminus single-letter codes) does not apply. Instead, its primary structure is defined by its covalent arrangement: a nicotinamide mononucleotide (NMN) moiety esterified to an adenosine monophosphate (AMP) moiety via a pyrophosphate linkage. Understanding this distinct nucleotide architecture is essential when designing enzymatic assays, ligand-binding studies, or chromatography methods where peptide separation protocols would yield invalid results.

Molecular Weight Considerations, Counterion Content, and Salt Form Analysis

In quantitative laboratory settings, applying the uncorrected bulk mass of a reagent to calculate molar concentration introduces significant analytical error. Lyophilized NAD+ is typically supplied as either a free acid or a mono/disodium salt. Furthermore, like synthetic peptides supplied as trifluoroacetate (TFA) or acetate salts, commercial coenzymes absorb atmospheric moisture (hygroscopic bound water) and contain variable counterion percentages.

The net chemical content (or net coenzyme content) reflects the actual percentage of active NAD+ (MW 663.43 g/mol) relative to the total mass, which includes counterions (such as Na+ at ~22.99 g/mol per ion) and residual hydration water. For instance, disodium NAD+ (C21H25N7Na2O14P2) exhibits a theoretical formula weight of approximately 707.40 g/mol. When preparing precise stock solutions for enzymatic kinetics, investigators must consult the batch-specific certificate of analysis to adjust mass measurements based on high-performance liquid chromatography (HPLC) purity and elemental sodium/water analysis.

To quickly recalculate target mass based on net purity percentages and target molar concentrations, researchers can utilize the online laboratory reconstitution calculator tool.

Physicochemical Properties and pH Stability Profiles

In vitro experimental design requires strict control over environmental parameters, as NAD+ displays distinct pH-dependent degradation pathways. In aqueous solution, the dinucleotide pyrophosphate backbone and the nicotinamide-riboside glycosidic bond are sensitive to both strongly acidic and strongly alkaline conditions.

In neutral to mildly acidic media (pH 3.0 to 6.0), aqueous NAD+ solutions demonstrate optimal thermodynamic stability at low temperatures (2–8°C or frozen at -20°C). However, exposure to alkaline conditions (pH > 7.5) accelerates the cleavage of the nicotinamide ring, generating fluorescent degradation products and reducing total coenzyme activity. Conversely, strongly acidic solutions (pH < 2.0) cause rapid hydrolysis of the purine nucleosidic linkage. Consequently, researchers preparing cell culture media or enzyme buffers should maintain pH monitoring and utilize non-nucleophilic buffers such as HEPES or Tris-HCl within a controlled pH range of 6.8 to 7.4.

Preclinical Mechanisms: Cellular Redox and Enzymatic Consumption Pathways

In preclinical model systems, NAD+ serves a dual role: it functions as a reversible hydride acceptor/donor in metabolic redox reactions and as a consumed cosubstrate in non-redox signaling cascades. In its oxidized state (NAD+), the pyridinium ring accepts two electrons and one proton from metabolic intermediates (catalyzed by dehydrogenases), reducing to NADH. This redox balance—expressed as the NAD+/NADH ratio—directly regulates glycolysis, the tricarboxylic acid (TCA) cycle, and mitochondrial oxidative phosphorylation.

Beyond metabolic electron transport, in vitro studies highlight NAD+ as an obligate substrate for three primary classes of regulatory enzymes:

1. Silent Information Regulator 2 (Sirtuin) Family: NAD+-dependent deacetylases (SIRT1–SIRT7) that cleave the nicotinamide moiety to transfer acetyl groups from target proteins to ADP-ribose, forming O-acetyl-ADP-ribose. 2. Poly(ADP-ribose) Polymerases (PARPs): Enzymes involved in DNA damage repair that consume NAD+ to synthesize branched PAR chains on target nuclear proteins. 3. Cyclic ADP-Ribose Synthases (CD38/CD157): Ecto-enzymes that convert NAD+ into secondary messengers such as cyclic ADP-ribose (cADPR) to regulate intracellular calcium mobilization.

Because these signaling pathways continually consume NAD+, maintaining accurate micro-molar concentrations in organoid or cell line models is critical for evaluating metabolic flux.

Analytical Verification: HPLC-UV and Mass Spectrometry Protocols

Validating the identity, molecular mass, and purity of NAD+ batches requires rigorous analytical methodologies. At PX1 Research, every lot undergoes rigorous quality control using high-performance liquid chromatography coupled with ultraviolet detection (HPLC-UV) and electrospray ionization mass spectrometry (ESI-MS).

• Reverse-Phase HPLC (RP-HPLC): Standard analytical runs utilize C18 stationary phases with ion-pairing reagents (such as triethylammonium acetate) or hydrophobic interaction chromatography (HIC) to resolve NAD+ from potential degradation products, including AMP, NMN, and nicotinamide. Absorbance is routinely monitored at 260 nm (peak adenine absorption). • Mass Spectrometry (ESI-MS): Negative-mode ESI-MS generates a prominent precursor ion [M-H]- at m/z 662.1, alongside double-charged species [M-2H]2- at m/z 330.5, confirming the intact dinucleotide mass without fragmented counterions.

Detailed analytical spectrum files and raw HPLC chromatograms are published directly in our open-access research library for laboratory verification.

Comparative Analysis: NAD+ and Related Metabolic Research Compounds

When designing preclinical protocols investigating mitochondrial efficiency, cellular aging, or energy homeostatic pathways, researchers frequently compare NAD+ with other small-molecule metabolites and mitochondrial-targeted peptides. Understanding the relative molecular weights, stability, and cellular uptake mechanisms of these reference compounds allows investigators to select the appropriate compound for specific assay types.

Unlike direct NAD+ supplementation in cell culture—where intact dinucleotide uptake across the plasma membrane can be rate-limited by ecto-enzyme activity—smaller precursors or peptide signaling molecules operate via distinct mechanisms. For instance, nicotinamide mononucleotide (NMN) has a lower molecular weight (~334.22 g/mol) and serves as an immediate enzymatic precursor. In peptide research, mitochondrial-derived peptides like MOTS-c (MW ~2174.6 g/mol) and cardiolipin-targeting tetrapeptides like SS-31 (MW ~639.8 g/mol) act on downstream mitochondrial energetics and ROS clearance rather than serving as direct coenzyme substrates. Researchers seeking to establish bulk ordering for comparative multi-compound studies can review our wholesale research portal for institution-level assay design.

Laboratory Handling, Reconstitution, and Storage Protocols

To preserve structural integrity and prevent premature hydrolysis, laboratory personnel must follow strict storage and handling guidelines upon receiving lyophilized NAD+:

1. Temperature Management: Dry powder should be stored at -20°C in a desiccated container protected from light. Under these conditions, the unconstituted dinucleotide remains stable for extended periods. 2. Reconstitution Protocol: Reconstitute using sterile, cold, deionized water or neutral, non-alkaline laboratory buffers (e.g., pH 6.5–7.0). Equilibrate the vial to room temperature prior to opening to minimize condensation uptake. 3. Aliquoting & Freezing: Reconstituted stock solutions should be divided into single-use research aliquots and stored at -80°C. Repeated freeze-thaw cycles must be avoided, as temperature fluctuations accelerate pyrophosphate cleavage.

PX1 Research Quality Assurance and Manufacturing Standards

PX1 Research maintains an uncompromising commitment to analytical rigor, supplying USA-manufactured research compounds designed exclusively for laboratory use. Every batch of NAD+ and secondary reference reagents is produced in facilities adhering to strict quality systems, ensuring consistent lot-to-lot reliability.

Our analytical testing process includes mandatory ISO 17025 accredited laboratory testing. Every lot undergoes full HPLC purity quantification, LC-MS identity confirmation, residual solvent determination, and bacterial endotoxin testing (LAL assay) to guarantee suitability for sensitive cell culture and enzyme assays. Products are dispatched directly from our California and Arizona fulfillment centers with same-day shipping (Monday–Friday) to support academic and commercial research timelines.