Structure elucidation of the thermal degradation products of the nucleotide cofactors NADH and NADPH by nano-ESI-FTICR-MS and HPLC-MS.
Redox cofactors like NADH and NADPH are essential for the catalytic activity of several oxidoreductases. Here, we describe a comparative study of the thermal degradation products of both cofactors in the dry and liquid states. The degradation products were first separated, detected, and quantified by high-performance liquid chromatography (HPLC). Subsequently, selected main fractions were investigated by nanoelectrospray ionization-Fourier transform ion cyclotron resonance mass spectrometry (MS). Additionally, HPLC-MS was used to elucidate the structure of all degradation products. From these data, degradation pathways for both the liquid and the solid states were elucidated. Thermal degradation in water is significantly faster compared to degradation in the solid state. Hydrolysis and oxidative ring opening of the reduced nicotinamide adenine dinucleotide (phosphate) were shown to be the main reaction paths. Surprisingly, no significant differences were observed between the degradation of both cofactors in solution and in the solid state. Our results demonstrate that the stability of both cofactors is not limiting at moderate temperatures if they are used in the dry state (e.g., solid/gas catalysis). Significant degradation of dry cofactors was only observed under conditions, which are usually not appropriate for biocatalysis (>95 °C). Besides, the situation is completely different in solution where degradation is already observed at moderate temperatures.
Hofmann
Diana
D
ZCH/BioSpec, Forschungszentrum Jülich GmbH, Leo-Brandt-Str., 52425 Jülich, Germany. d.hofmann@fz-juelich.de
Wirtz
Astrid
A
Santiago-Schübel
Beatrix
B
Disko
Ulrich
U
Pohl
Martina
M
D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D005583:Fourier Analysis; D006358:Hot Temperature; D009243:NAD / Q000737:chemistry*; D009249:NADP / Q000737:chemistry*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
D009243:NAD; D009249:NADP