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 usin g changes in the UV-visible absorption spectra of these compounds. The spectra of cofactor (reduced form) were monitored at 340 nm wavelengt h in different buffers, showing a faster degradation in phosphate buff er. This was assigned to the adduct formation between phosphate and NA DH (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 pre pared aqueous solutions of coenzyme were analyzed after 40 min under t he 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 eff ects. Therefore, it was verified that the better conditions for cofact or use were alkaline media employing Pipes buffer or its derivatives a nd low temperatures. (C) 1998 Academic Press.