ASSOCIATIONS OF BETA NERVE GROWTH FACTOR WITH BOVINE SERUM ALBUMIN AS WELL AS WITH THE ALPHA AND GAMMA SUBUNITS OF THE 7S MACROMOLECULE

Abstract— The association of ¹²⁵ I‐diiodo‐Beta nerve growth factor with bovine serum albumin (BSA), as well as with the Gamma and Alpha subunits of the 7S nerve‐growth factor (NGF) macromolecule are described. In the absence of added protein, the stable thermodynamic state for Beta (pH 7.4) at concentrations below 10⁻⁷ M is absorbed to the walls of the reaction vessel. Above 10⁻⁷ M the sites on the wall of the reaction vessel begin to saturate, and Beta is found free in solution. Addition of other soluble proteins to the system (i.e. BSA, Alpha and Gamma) causes a displacement of the Beta from the walls of the reaction vessel. This displacement is the result of the binding of the Beta to the added soluble protein. The binding of Beta to BSA is a complex function of BSA concentration, suggesting multiple sets of sites and/or cooperative interactions. In contrast, the characteristics of the association of Beta with Gamma and Alpha indicate an interaction between discrete sets of sites on the respective polypeptide chains. The Beta‐Gama association is a bimolecular association with an apparent first thermodynamic association constant of 6.25 × 10⁵M⁻¹. The association between Beta and Alpha is also bimolecular and has an apparent first thermodynamic association constant of 2.0 × 10⁵M⁻¹. In addition, dissociation studies suggest that the Gamma‐Beta complex binds Alpha with a substantially higher affinity than does Beta or Gamma alone. These data strongly support the conclusion that there is a unique biologically determined relationship among these polypeptides. The data are discussed with respect to the experimental use of Beta NGF. A new operational paradigm for the controlled use of the Beta NGF is presented. This approach is based on the use of a thermodynamically stabilized 7S complex and the analysis of relative apparent affinities.