The software program, HINT (Hydropathic INTeractions), which characterizes non-polar-non-polar, polar-polar, and non-polar-polar interactions, has been used to examine subunit interface associations involved in the hemoglobin allosteric transition at a residue and atomic level. HINT differs from many other computational programs in that it is based not on a statistical method or a force-field but employs parameters experimentally determined from solvent transfer experiments. The main focus of this study is to compare HINT scores that are based upon experimentally and thermodynamically derived measurements with experimentally determined thermodynamic results. The HINT analysis yields a good first-order approximation of experimentally measured energies for these interactions as determined by free energies of dimer-tetramer assembly for mutant hemoglobins. The results provide a framework for understanding subunit stabilities based upon individual atom interactions and repulsions. HINT, in agreement with previous analyses, indicates that: (1) the alpha 1 beta 1 and alpha 2 beta 2 subunit contacts are stabilized via several polar and many hydrophobic interactions with few repulsive contact areas in both the T (deoxyhemoglobin) and R (oxyhemoglobin) structures; (2) the alpha 1 alpha 2 subunit contacts are primarily stabilized by polar salt bridge linkages in both T and R states; and (3) the alpha 1 beta 2 and alpha 2 beta 1 contacts have both strong positive and negative interactions in both T and R states with few hydrophobic interactions. The HINT scoring methodology provides a quantitative characterization of the major role of the alpha 1 beta 2 and alpha 2 beta 1 interfaces in the T --> R quaternary transition. HINT also confirms the stronger hydrogen bond formation in mutant Kb Rothschild (Trp 37 beta --> Arg) with Asp94 alpha 1 that gives rise to a low-affinity (deoxy) hemoglobin. HINT shows that the stabilization of the alpha 1 beta 2 interface with mutant Hb Ypsi-lanti (Asp99 alpha --> Tyr) produces a high-affinity (oxy) hemoglobin by reducing hydrophobic-polar contacts in the R state. HINT interaction maps also identified specific sites for mutagenesis at the alpha 1 beta 2 interface that can be explored to shift the allosteric equilibrium in either direction. In addition, the HINT program provides useful diagnostic data for checking the quality of refined crystallographic structures. (C) 1997 Academic Press Limited.