The 3D HNHB experiment allows to measure J(N-HB) coupling constants of ¹⁵N-labeled proteins in H₂O using the so-called quantitative-J correlation approach. In addition, J(N-HA) coupling constants can be also measured.

Implementation on AVANCE spectrometers equipped with pulsed field gradients (PFGs) is feasible.

The 3D HNHB pulse sequence has been proposed in 97JB301 which allows the measurement of the coupling constants directly from the spectrum. However, the original 2D versions were proposed some years ago ( 91JMR636 and 93JB239 ) and a separate 2D reference experiment needs to be recorded to extract coupling constant values.

The spectra affords two types of signals:

Diagonal signals at (F1,F2,F3)=(N,NH,NH)

Cross-peak signals at (F1,F2,F3)=(N,HB,NH)

The intensity ratio between the cross-peak and the diagonal peak provides a direct measure for the magnitude of J(N-HB)