Phenolic ligands, e.g., phenol and m-cresol, bind to 2Zn(II)-insulin h
examers and induce a conformational change at the N-terminus of the B-
chain for each monomer. The binding of these phenolic ligands to 2Zn(I
I)-insulin hexamers has been studied by isothermal titrating calorimet
ry (ITC). The binding isotherms were modeled and thermodynamic paramet
ers were quantified using a novel, flexible algorithm that permitted t
he development of a hierarchical series of physical models. With the i
nsulin hexamer represented as a dimer of trimers, the modeling demonst
rated that ligand binding is highly cooperative in nature, both intra-
and inter-trimer. The isotropic inter-trimer cooperativity was domina
nt and negative in every system studied, with initial binding constant
s typically an order of magnitude greater for the binding of ligands t
o the first trimer relative to the second. The inter-trimer cooperativ
ity estimated from the modeling of solution calorimetry data is consis
tent with a T-6 <-> T(3)R(3) <-> R(6) equilibrium first proposed from
crystallographic investigations. Intra-trimer cooperativity was presen
t only in the enthalpy coefficient space, not in the equilibrium coeff
icient space, and therefore, less of a factor. The order of binding af
finity for the ligands studied is resorcinol much greater than phenol
greater than or equal to m-cresol as determined from their overall fre
e energies of binding to the 2Zn(II)-insulin hexamer (-26.6, -23.4, an
d -23.4 kcal/mol, respectively) and their intrinsic binding constants
(8780, 5040, and 3370 L/mol, respectively) at 14 degrees C. The temper
ature dependence of phenol binding to 2Zn(II)-insulin hexamer was mode
led. Increasing temperature decreased the magnitude of both the intrin
sic binding constant and the inter-trimer cooperativity. The second ph
ase of the ITC binding profile was also found to be highly temperature
dependent, At lower temperatures the second phase is endothermic but
gradually decreases with increasing temperature and subsequently becom
es exothermic. This effect is attributed to loss of water from the hyd
ration shell of the insulin hexamer with increasing temperature and co
nsequently reduces the entropic contributions to the T <-> R transitio
n in the phenol/2Zn(II)-insulin hexamer system.