PRACTICAL CONSIDERATIONS FOR THE MEASUREMENT OF THE HOMONUCLEAR OVERHAUSER EFFECT ON STRONGLY RELAXED PROTONS IN PARAMAGNETIC PROTEINS

Experimental and interpretive problems are considered in the application of nuclear Overhauser effect, measurements on strongly relaxed resonances in paramagnetic metalloproteins. Solutions to the Bloch equations under high-power irradiation show that off-resonance saturation is severe over a considerable spectral width and exceeds the expected NOEs unless the degree of on-resonance saturation is restricted to well below 100%. Semi-quantitative simulations reveal excellent correlation between predicted and observed off-resonance effects. It is shown, moreover, that the decoupler power necessary to validate the use of the standard truncated NOE, TOE, equations are not experimentally accessible for strongly relaxed lines. Hence, time-dependent NOE data for strongly relaxed lines require the use of more complete equations that explicitly consider the rate and degree of on-resonance saturation. Attempts to 6t time-dependent NOE data to the simple TOE equations based on assuming instantaneous and complete on-resonance saturation are shown to lead to substantial errors in intrinsic and cross-relaxation rates. It is demonstrated for a pair of high-spin ferric met-myoglobins that true NOES and off resonance effects can be differentiated in steady-state NOE experiments, and the former semiquantitated, by analyzing either the effect of stepping the decoupler through a resonance or, in a crowded spectral window, the effect on all lines upon constant-power saturation of each component resonance. © 1991.