Figure 2.

³¹P chemical exchange analysis for 30 mM NADH in pure deuterated solvents of DMSO-d₆ and D₂O. (A) Investigation of the irradiation power dependence on ³¹P CEST line widths, normalized by the square root of the spin–spin relaxation rate (FWHM/), at temperatures of 310, 323, 333, and 343 K in pure D₂O. The increasing slope observed at these temperatures signifies an enhancement in the chemical exchange rate with increasing temperature. (B) A parallel analysis conducted in pure DMSO-d₆. The uniform slopes across the studied temperatures indicate a negligible chemical exchange in this solvent. (C) Variation in the ratio of the absolute integral of the ³¹P signal from 1D NMR spectra in 30 mM NADH solutions, using binary mixed solvents of DMSO-d₆ and D₂O measured from 298 K to 343 K and adjusted for the temperature dependence as discussed in the main text.. An increasing ratio with a higher fraction of DMSO-d₆ suggests that the presence of DMSO-d₆ reduces the “dark” population of NADH in the solution.