The synthesis of 15N- and deuterium-substituted, spin-labeled analogues of NAD+ and their use in EPR studies of dehydrogenases.

Two spin-labeled analogues of NAD+ were synthesized with an 15N and perdeuterated nitroxide radical, 4-amino-2,2,6,6-[2H17, 15N]tetramethylpiperidone-1-oxyl, which was attached to either the C-6 or C-8 position of the purine ring. The EPR spectra of these derivatives exhibit an approx. 6-fold increase in sensitivity compared with the corresponding 14N, protonated analogues due to a decrease in both the number of nuclear manifolds (from three to two) and the linewidth. The enhanced spectral resolution obtained with (2H17, 15N)spin-labeled-NAD+ analogues has facilitated simulation of the EPR lineshape of the nucleotide bound to lactate dehydrogenase (L-lactate:NAD+ oxidoreductase, EC 1.1.1.27). The spin-label moiety exhibits highly constrained motion indicative of a single environment. The motion of the spin label does not reflect the overall motion of the enzyme; rather, it is characteristic of some limited mobility relative to the lactate dehydrogenase. By contrast, the spin label on the membrane-bound enzyme, D-beta-hydroxybutyrate dehydrogenase (D-beta-hydroxybutyrate:NAD+ oxidoreductase, EC 1.1.1.30), is completely immobilized and exhibits two distinct spectral components for spin-labeled NAD+, which appear to differ in the polarity of the environment of the nitroxide.