What Is NAD+? A Dinucleotide, Not a Peptide

NAD+ (β-nicotinamide adenine dinucleotide) is a dinucleotide: two nucleotides, nicotinamide mononucleotide and adenosine monophosphate, joined through their phosphate groups by a pyrophosphate bridge. It is not a peptide, because it contains no amino acids and no peptide bonds. Its molecular formula is C21H27N7O14P2, its average molecular weight is 663.43 g/mol and its CAS number is 53-84-9.

The "+" in the name marks a positively charged nitrogen atom in the nicotinamide ring. That ring is also where the molecule's best-known chemistry happens: a reversible two-electron reduction to NADH. This page covers NAD+ as a molecule: how it is built, what its formula and mass are, how the NAD+/NADH pair behaves chemically, and how the material is analysed and stored.

Is NAD+ a peptide?

No. NAD+ is often listed next to research peptides, and our catalogue places it in the same research area as two peptides, but it belongs to a different chemical class. A peptide is a chain of amino acids joined by amide (peptide) bonds, as explained in what are peptides. NAD+ is built from the same kinds of units as RNA: nitrogenous bases, the sugar D-ribose and phosphate.

Phosphorus is a quick way to tell the two classes apart on paper. A standard peptide formula contains none, whereas C21H27N7O14P2 contains two phosphorus atoms.

The structure of NAD+, piece by piece

NAD+ contains five smaller units. Two are bases: nicotinamide, the amide of pyridine-3-carboxylic acid, and adenine, a purine. Each base is bonded through a ring nitrogen to carbon 1 of a D-ribose ring, and each ribose carries a phosphate on its carbon 5.

Nicotinamide, ribose and phosphate make one nucleotide, nicotinamide mononucleotide (NMN). Adenine, ribose and phosphate make the other, adenosine monophosphate (AMP). Joining the two phosphate groups by condensation, with loss of water, forms the pyrophosphate bridge, and two nucleotides joined together make a dinucleotide. The linkage is unlike the one between neighbouring nucleotides in RNA, where a single phosphate joins carbon 3′ of one ribose to carbon 5′ of the next. In NAD+ the two halves are joined 5′ to 5′ through two phosphates.

PubChem's systematic name spells out the same arrangement: two oxolane (ribose) rings, both configured (2R,3S,4R,5R), one carrying 6-aminopurin-9-yl (adenine) and the other 3-carbamoylpyridin-1-ium-1-yl (nicotinamide), connected through the two phosphoryl groups.

What the "β" and the "+" mean

The "β" in β-NAD+ describes the configuration at carbon 1 of the ribose that carries nicotinamide: the bond to the pyridine ring lies on the same face of the sugar ring as the CH2–O group at carbon 4. The α-anomer is a different stereoisomer with the same formula and mass, so "β" is part of the identity of the material rather than a grade.

The "+" refers to a single atom. The nitrogen of the pyridine ring uses its lone pair to bond to ribose, so it carries a formal positive charge, which makes the ring a pyridinium ion. The sign does not give the charge of the whole molecule. The formula C21H27N7O14P2 describes an inner salt, in which one ionised phosphate balances the pyridinium charge, and the formal name on a reagent supplier's data sheet ends with the words "inner salt". In water near neutral pH the second phosphate is ionised as well, so the molecule as a whole carries a net charge of −1.

Formula, mass and salt form

The formula C21H27N7O14P2 gives two masses, and each has its own job. The average molecular weight, 663.43 g/mol, uses standard atomic weights and is the figure for weighing and for converting mass to moles. The monoisotopic mass, 663.1091 Da, uses the most abundant isotope of each element and is the figure a mass spectrometer measures.

Both values describe the free acid, written as the inner salt. NAD+ can also be isolated as salts, such as sodium salts, and a salt has a different formula and molecular weight. A certificate or specification should therefore state which form the material is, because the conversion from a weighed mass to an amount in moles depends on it.

NAD+ and NADH: a redox pair

NAD+ is the oxidised member of a redox couple. Its reduced partner, NADH, differs by two electrons and one proton, which arrive together on the nicotinamide ring as a hydride ion (H−):

NAD+ + H+ + 2 e− ⇌ NADH

The hydride adds to carbon 4 of the pyridinium ring, the position opposite the ring nitrogen. The ring loses its positive charge and its aromaticity and becomes a 1,4-dihydropyridine. PubChem lists NADH as C21H29N7O14P2 (665.4 g/mol), two hydrogen atoms more than NAD+ in the neutral forms both are written in. In enzyme assays in vitro, dehydrogenases catalyse this hydride transfer between a substrate and NAD+, which is why NAD+ is classed as a coenzyme. The reaction itself is a property of the nicotinamide ring.

The change in the ring is easy to follow with a spectrophotometer:

• Near 260 nm, both forms absorb. This band comes from the adenine ring, which is the same in NAD+ and NADH. A reagent supplier's data sheet for the free acid gives absorption maxima at 207 and 260 nm.
• Near 340 nm, only NADH absorbs. The 1,4-dihydropyridine ring of NADH has an extra band here that the pyridinium ring of NAD+ lacks. Horecker and Kornberg reported absorption coefficients for this "reduced band" in 1948, and it remains the standard signal for following the NAD+/NADH couple in vitro.

The two forms also differ in chemical stability. In their study of pyridine nucleotide stability, Lowry, Passonneau and Rock (1961) reported that the oxidised forms withstand acid but decompose in alkali, while the reduced forms decompose in acid and withstand alkali. Analytical methods therefore handle NAD+ and NADH differently, and a method built for one form is not automatically suitable for the other.

How NAD+ is analysed

Both ions come from the monoisotopic mass: plus one proton (1.00728 Da) in positive mode, minus one proton in negative mode. NADH sits about 2.016 mass units higher, at m/z 666.132 in positive mode. NAD+ has a natural isotope peak close to that position, so NADH shows up as a peak larger than the isotope pattern predicts, or as a separate peak at high resolving power. More detail on the two main methods is in the guides to HPLC purity and mass spectrometry for identity. Both are written for peptides, but the principles carry over.

For our NAD+, the purity specification is ≥99% (HPLC, supplier specification), and certificate status is shown on every product page. When reading any certificate for NAD+, check that the formula and molecular weight match the form stated (free acid or a salt), that the observed mass matches the values above, that the HPLC method gives its detection wavelength, and that the batch number and analysis date match the vial. The general layout of such a document is covered in how to read a certificate of analysis.

Storing a sealed vial of NAD+

NAD+ is supplied as a lyophilised powder in a sealed vial. Store the sealed vial at 2–8 °C for short-term storage, or at −20 °C and below for long-term storage. Protect it from light and avoid repeated freeze-thaw cycles. A reagent supplier's data sheet for the free acid gives −20 °C as its storage temperature.

Because the solid is highly hygroscopic, keeping the vial closed matters as much as the temperature. A cold vial opened in room air collects condensation, and any water the powder takes up adds to its gross mass, which changes how much NAD+ a weighed amount contains. The same principles for sealed lyophilised solids are set out in storing lyophilised peptides; they apply to NAD+ even though it is not a peptide.

NAD+ belongs to the Mitochondrial Compounds research area, where it is catalogued with two peptides of different design: MOTS-c, a linear sixteen-residue peptide, and SS-31, an aromatic-cationic tetrapeptide. Chemical terms used on this page are collected in the glossary.