Properties of Human Prolactin (PRL) and H27A-PRL, a Mutant That Does Not Bind Zn ++

The ability of protein hormones to self-associate is likely to play an important role in concentrating hormones into secretory granules; therefore, the aggregation properties of human PRL and H27A mutant were investigated. Human PRL bound ⁶⁵Zn⁺⁺; the Scatchard analysis was convex up, and limited by the solubility of PRL, but at least 0.7 mole Zn⁺⁺ bound per mole of PRL. Binding of ⁶⁵Zn⁺⁺ to H27A-PRL was greatly reduced. The biological activity in an Nb2 cell assay and the circular dichroism spectrum of wild type and H27A-PRL were similar, indicating the H27A mutant folded correctly, and the binding of Zn⁺⁺ to the high affinity site is not essential for biological activity. Dynamic light scattering measurements indicated 10 and 20 μM Zn⁺⁺ caused some aggregation of both wild type and H27A-PRL. Sedimentation equilibrium analysis indicated that PRL is primarily a monomer in the absence of Zn⁺⁺ and that there is increasing self-association in the presence of 5 and 10 μM Zn⁺⁺. The mutant H27A exhibited a greater tendency to aggregate without changing detectably the mode of association. Although human PRL binds Zn⁺⁺ as human GH does, it differs in that the ability to bind Zn⁺⁺ and to self-associate were decoupled in PRL. Human PRL must have two types of interactions with Zn⁺⁺; one is binding to a site involving histidine 27, and the other is weaker interactions that induce self-association of PRL.