The 3D HNHA experiment allows to measure J(HN-HA) coupling constants of ¹⁵N-labeled proteins in H₂O using the so-called quantitative-J correlation approach.

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

The original 3D HNHA pulse sequence was proposed in 93JACS7772 . Several modified pulse sequences have been reported:

Incorporation of the WATERGATE scheme to improve water suppression ( 97JB263 ).

Non-purged version in which the 1H 90 degree puge pulse inmediately prior to acquisition has been removed ( 94JB193 ). Improved sensitivty is obtained although quantitative measurement becomes difficult.

94JB871

N,C-filtered version to measure J(HN-HA)coupling constants in unlabeled peptides complexed with labeled proteins ( 01JB23-20)

The spectra affords two types of signals:

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

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

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

General search for HNHA