Nicotinamide Adenine Dinucleotide - Physical and Chemical Properties

Physical and Chemical Properties

Nicotinamide adenine dinucleotide, like all dinucleotides, consists of two nucleotides joined by a pair of bridging phosphate groups. The nucleotides consist of ribose rings, one with adenine attached to the first carbon atom (the 1' position) and the other with nicotinamide at this position. The nicotinamide moiety can be attached in two orientations to this anomeric carbon atom. Because of these two possible structures, the compound exists as two diastereomers. It is the β-nicotinamide diastereomer of NAD+ that is found in organisms. These nucleotides are joined together by a bridge of two phosphate groups through the 5' carbons.

In metabolism, the compound accepts or donates electrons in redox reactions. Such reactions (summarized in formula below) involve the removal of two hydrogen atoms from the reactant (R), in the form of a hydride ion (H−), and a proton (H+). The proton is released into solution, while the reductant RH₂ is oxidized and NAD+ reduced to NADH by transfer of the hydride to the nicotinamide ring.

RH₂ + NAD+ → NADH + H+ + R;

From the hydride electron pair, one electron is transferred to the positively charged nitrogen of the nicotinamide ring of NAD+, and the second hydrogen atom transferred to the C4 carbon atom opposite this nitrogen. The midpoint potential of the NAD+/NADH redox pair is −0.32 volts, which makes NADH a strong reducing agent. The reaction is easily reversible, when NADH reduces another molecule and is re-oxidized to NAD+. This means the coenzyme can continuously cycle between the NAD+ and NADH forms without being consumed.

In appearance, all forms of this coenzyme are white amorphous powders that are hygroscopic and highly water-soluble. The solids are stable if stored dry and in the dark. Solutions of NAD+ are colorless and stable for about a week at 4 °C and neutral pH, but decompose rapidly in acids or alkalis. Upon decomposition, they form products that are enzyme inhibitors.

Both NAD+ and NADH strongly absorb ultraviolet light because of the adenine. For example, peak absorption of NAD+ is at a wavelength of 259 nanometers (nm), with an extinction coefficient of 16,900 M−1cm−1. NADH also absorbs at higher wavelengths, with a second peak in UV absorption at 339 nm with an extinction coefficient of 6,220 M−1cm−1. This difference in the ultraviolet absorption spectra between the oxidized and reduced forms of the coenzymes at higher wavelengths makes it simple to measure the conversion of one to another in enzyme assays – by measuring the amount of UV absorption at 340 nm using a spectrophotometer.

NAD+ and NADH also differ in their fluorescence. NADH in solution has an emission peak at 460 nm and a fluorescence lifetime of 0.4 nanoseconds, while the oxidized form of the coenzyme does not fluoresce. The properties of the fluorescence signal changes when NADH binds to proteins, so these changes can be used to measure dissociation constants, which are useful in the study of enzyme kinetics. These changes in fluorescence are also used to measure changes in the redox state of living cells, through fluorescence microscopy.