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We have obtained an experimental estimate of the free energy change associated with variations at the interface between protein subunits, a subject that has raised considerable interest since the concept of accessible surface area was introduced by Lee This paper deals with the experimental determination of the free energy change associated with modification of the surface buried in a protein-protein contact, a point of general interest in understanding stability and recognition in proteins. Since the pioneering work of Lee and Richards (1), a wealth of experimental and theoretical investigations have attempted to understand and quantify the forces stabilizing protein oligomers and complexes. After Chothia (2, 3) drew attention to the significance of the surface buried at an interface, other investigators emphasized shape complementarity (4), residue propensities (5), and the relative contribution of polar interactions and hydrophobic effects to the overall ⌬G 0 in proteinprotein association (6-8). Experimental studies have used different proteins to understand the effect of a point mutation on enzyme-inhibitor (9, 10) or antibody-antigen complexes (11-13), as well as subunit interactions in mutants of human Hb (14). These studies led to the conclusion that both types of weak interactions play a role, and that Atom Solvation Parameters derived from partition coefficients can be used to evaluate the stabilization energy at protein interfaces (8,(15)(16)(17).Questions arise whether the theoretical studies can be a guideline in the analysis and design of experiments with a resolution beyond the qualitative prediction of results and how far values obtained from physico-chemical considerations can be useful in protein engineering by mutagenesis. Although it is recognized that buried hydrophobic surface contributes to the stability of a protein-protein contact, theoretical estimates of the free energy gain associated with burial of 1 Å 2 of hydrophobic surface are variable and range between 4 and 32.5 cal͞mol (see ref. 16 for a recent review). We have addressed this point experimentally by using the dimer-tetramer association of human Hb mutants.By analytical ultracentrifuge, we have determined the equilibrium constant for the 2␣␤ 3 (␣␤) 2 association for five single and three double mutants of human HbCO. The mutations are all at the ␣ 1 ␤ 2 interface (Fig. 1) except one at the ␣ 1 ␤ 1 interface, which has a larger number of contacts and is unchanged in the T 3 R allosteric transition (14,(18)(19)(20). The changes in association free energy relative to HbA have been correlated with changes in the buried surface area due to mutations, to dissect the contribution of the polar and hydrophobic contacts in the stabilization of the tetramer. Taking a ϩ1.5 kcal͞mol complementarity cost associated with each mutation, we found that the ⌬(⌬G MATERIALS AND METHODSSite-directed mutants of HbA were produced in Escherichia coli and were purified as described (21,22). Reagents were of analytical grade. Ultracentrifuge ex...

A procedure for 3,4-dihydroxyphenylalanine decarboxylase from pig kidney purification is described in detail.The preparation has no detectable impurity on electrophoresis and on ultracentrifugation and has a coenzyme content and a specific activity comparable with the same enzyme purified by other authors.However two significant differences are observed: a different stimulation of activity by added pyridoxal 5'-phosphate and a nearly complete decarboxylation of ~-3,4-dihydroxyphenylalanine in absence of added coenzyme.Absorption, fluorescence and circular dichroism properties of the coenzyme-apoenzyme interaction are also described.The results are consistent with the existence of at least four coenzyme-apoenzyme complexes, three of them active.

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