Relation between Conformation and Association State

Abstract

Abstract The concentration-dependent equilibrium of glucagon in mildly alkaline solution involves the formation of associated forms of higher helicity. The equilibrium may be followed by the change of optical rotation with concentration. In acid solution, the effect is still present but much diminished. The association is accompanied by the generation of a new Cotton effect at 298 mµ, which is attributed to the single tryptophan residue. A further consequence of the association is a shift in the spectrophotometric titration curve of the tyrosine residues. Both at high and at low glucagon concentration, the ionization curves are fitted by two pKa values, which are 9.9 and 10.7 for the disaggregated material and 10.1 and 10.7 for the associated form. From the sequence, it is possible to deduce that it is Tyr-13 which undergoes a pKa shift on association, whereas the remaining tyrosine, Tyr-10, seems to be unaffected. It is suggested that the association should be viewed in terms of two linked equilibria, the associated state acting as a thermodynamic trap for the structured conformation. It is shown that the true association constants must differ from the apparent ones by a factor which may correspond to several kilocalories per mole in free energy. It is suggested that such a mechanism forms a general basis for interrelations between aggregation state and conformation and is relevant to such phenomena as refolding pathways of denatured proteins.