Study of NADH Stability Using Ultraviolet–Visible Spectrophotometric Analysis and Factorial Design

The chemical stability of nicotinamide adenine dinucleotide coenzyme (NADH/NAD+) and its derivatives (NADPH/NADP+) was investigated using changes in the UV-visible absorption spectra of these compounds. The spectra of cofactor (reduced form) were monitored at 340 nm wavelength in different buffers, showing a faster degradation in phosphate buffer. This was assigned to the adduct formation between phosphate and NADH (pyridine ring). The three-factor-two-level factorial design study evaluated the contributions: buffers (phosphate and Pipes 0.1 M), pH's (6.8 and 7.8), and temperature (25 and 30 degrees C). The freshly prepared aqueous solutions of coenzyme were analyzed after 40 min under the different conditions (eight assays in triplicate) of the experiment. The main observed effects of the NADH oxidation were, in increasing order, pH, temperature, and buffer without appreciable interactive effects. Therefore, it was verified that the better conditions for cofactor use were alkaline media employing Pipes buffer or its derivatives and low temperatures.