Biochemical properties and binding parameters of the pancreatic estradiol binder

Because the estradiol binder is distributed throughout subcellular fractions from homogenates of the pancreas, an acetone powder was used in extracting soluble components from the entire tissue. The estradiol binder, obtained from the acetone powder of a bovine pancreas, was pH dependent, denatured by heat, inhibited by CuSO4 and reversibly denatured by urea. Binding was enhanced by Mg+2, a common enzymatic cofactor. Its molecular weight was greater than 66,000 D, the molecular weight of bovine serum albumin. An 11-fold purification resulted in a protein preparation that showed a saturating binding curve in equilibrium dialysis. Klotz has severely criticized, if not discredited, the hypothesis that a binding curve is typically due to two classical binding sites and is resolvable into two straight lines. We, therefore, determined the parameters of the binding curve by a computer analysis based on Wilkinson's algebraic method and on McLaren's proposal of surface relationships in enzymatic reactions. The inference from McLaren (namely, binding sites of a single affinity located on the surface of the macromolecular binder), offered a simpler explanation for curvature in Scatchard plots than the two affinity site or the varying affinity hypotheses. The analysis was also applied to other examples from the literature.