Iron binding to conalbumin. Calorimetric evidence for two distinct species with one bound iron atom.

When thermal denaturation of conalbumin solutions partially saturated with Fe(III) is observed by differential scanning calorimetry, four endotherms are observed between 40 and 100 degrees. The relative size of these four endotherms is determined by the Fe(III) to conalbumin ration. At a heating rate of 10 degrees/min, in Tris buffer at pH 7.5, observed endotherm temperature maxima and enthalpies of denaturation are: conalbumin, 63 degrees, 320 kcal/mol; intermediate I, 68 degrees, intermediate I, 77 degrees; Fe2-conalbumin, 84 degrees, 630 kcal/mol. These four endotherms are observed over a range of protein concentration from 7 to 100 mg/ml and are unchanged when excess bicarbonate is present. Stoichiometric calculations of both total protein and total iron indicate that each intermediate endotherm results from denaturation of conalbumin molecules containing only one ferric ion. These experimental results are thus consistent with the presence of two different monomeric one-iron conalbumin intermediates. They strongly suggest that the two iron binding sites of conalbumin are not equivalent.