Weak and transient protein–protein interactions underlie numerous biological processes. However,
the location of the interaction sites of the specific complexes and the effect of transient,
non-specific protein–protein interactions often remain elusive. We have investigated the weak selfassociation
of human growth hormone (hGH, KD = 0.90 ± 0.03 mM) at neutral pH by the paramagnetic
relaxation enhancement (PRE) of the amide protons induced by the soluble paramagnetic
relaxation agent, gadodiamide (Gd(DTPA-BMA)). Primarily, it was found that the PREs are in
agreement with the general Hwang-Freed model for relaxation by translational diffusion (J. Chem.
Phys. 1975, 63, 4017–4025), only if crowding effects on the diffusion in the protein solution are
taken into account. Secondly, by measuring the PREs of the hGH amide protons at increasing hGH
concentrations and a constant concentration of the relaxation agent, it is shown that a distinction
can be made between residues that are affected only by transient, non-specific protein–protein interactions
and residues that are involved in specific protein-protein associations. Thus, the PREs of
the former residues increase linearly with the hGH concentration in the entire concentration range
because of a reduction of the diffusion caused by the transient, non-specific protein-protein interactions,
while the PREs of the latter residues increase only at the lower hGH concentrations but
decrease at the higher concentrations because of specific protein-protein associations that impede
the access of gadodiamide to the residues of the interaction surface. Finally, it is found that the
ultra-weak aggregation of hGH involves several interaction sites that are located in patches covering
a large part of the protein surface.