There are kits that can do this identification but they don't work when these co-factors are bound to enzymes. By using fluorescence, I have already identified a peak that could be one of those two or both. Thank you.
The absorbance maximum for NADPH and NADH is 340 nm. If you have enough of your protein to take an absorbance spectrum and you see a peak at that wavelength, then you probably have one of these nucleotides bound. The fluorescence emission maximum wavelength is 460 nm, so you can also look for that. Both of these wavelengths are well-separated from the aromatic amino acid absorbance peak at ~280 nm and the tryptophan fluorescence peak at 330-360 nm.
Another method is to denature the protein and analyze for released nucleotides by HPLC or mass spectrometry. Absorbance and fluorescence spectroscopy can also be done on the released nucleotides. You could also use one of the kits you mentioned on the released nucleotides.
May be you can try using Raman spectroscopy/SERS. Please see attached document
The absorbance maximum for NADPH and NADH is 340 nm. If you have enough of your protein to take an absorbance spectrum and you see a peak at that wavelength, then you probably have one of these nucleotides bound. The fluorescence emission maximum wavelength is 460 nm, so you can also look for that. Both of these wavelengths are well-separated from the aromatic amino acid absorbance peak at ~280 nm and the tryptophan fluorescence peak at 330-360 nm.
Another method is to denature the protein and analyze for released nucleotides by HPLC or mass spectrometry. Absorbance and fluorescence spectroscopy can also be done on the released nucleotides. You could also use one of the kits you mentioned on the released nucleotides.
The best way would be to denature the proteins using selective acid/base destruction followed by quantification of oxidized and reduced forms respectively. See the following reference for guidelines. Preller A, Guixé V, Ureta T. In vivo operation of the pentose phosphate pathway in frog oocytes is limited by NADP+ availability. FEBS Lett. 1999 Mar 5;446(1):149-52. PubMed PMID: 10100632. You should also do recovery caculations too to see how much NAD(P)H is lost during the extraction - spike duplicate extracts with known amounts of NAD(P)(H) and compare this to the theoretical recovery.
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