On the relative location of the inhibitor-and calcium-binding sites in bovine trypsin as determined by nuclear magnetic resonance. Possible ambiguities in paramagnetic probe mapping studies.

Possible pitfalls in mapping studies utilizing the nuclear relaxation rates induced by paramagnetic probes are pointed out. In cases in which a distance is sought between a paramagnetic ion and a small molecule (e.g. substrate, inhibitor, etc.), both bound non-covalently to a macromolecule, heterogeneity in the system with respect to the binding of either of them may result in ambiguous conclusions. It is shown that the trypsin-gadolinium (III)-inhibitor system is heterogeneous, as revealed in the dependence of the water and inhibitor proton line-widths upon both the Gd3+ and the enzyme concentrations and in the effects of added Ca2+ on the line-widths. The results imply that in published work (Abbott et al. (1975) Biochemistry 14, 4935) the distance from a weak rather than from the strong metal ion binding site of trypsin (EC 3.4.21.4) may have been determined.