Steroid-protein interactions. 36. The pH dependence of progesterone interaction with progesterone-binding globulin. Kinetic and equilibrium studies

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Abstract
The kinetics of binding and dissociation for the [guinea pig serum] progesterone-binding globulin (PBG)-progesterone complex were measured as a function of pH. The association rate constant appears to be independent of pH from pH 5-10 with an average value kon [association constant] = 8.5 .times. 107 M-1 s-1. The Kd is strongly pH dependent, with the dependency defined by: koff = ko (1 + [H+]/K1 + K2/[H+](1 + K3*/[H+])/(1 + K3/[H+]) [K1 and K2 are the Kd for 2 amphoteric groups; K3 and K3* are the Kd for a 3rd group, with K3 corresponding to the PBG-progesterone complex and K3* to free PBG]. The best values for the various parameters were ko = 0.0785 s-1, pK1 = 5.30, pK2 = 10.54, pK3* = 7.41, and pK3 = 7.21. Simpler expressions were inadequate to fit the data, and at least 3 ionizing residues are responsible for the stability of the PBG-progesterone complex. The affinity constant was determined by equilibrium dialysis over the range of pH 3-12. The ratio of the association and dissociation rate constants is in agreement with the affinity constant from pH 6.5-10.5. The influence of pH on the conformation and binding activity of PBG was also investigated. Denaturation by acid, base or guanidine hydrochloride leads to a reversible loss of binding activity. Regain of binding activity in all cases is slow with half-times of 0.5-2.7 h, depending on conditions. The rate of acid denaturation was determined as a function of pH. The protein was incompletely protonated at pH 1.4, suggesting a buried carboxylic acid residue. The slow renaturation of PBG might be due to the difficulty of burying a charged residue in the protein''s interior coupled with steric hindrance by the large carbohydrate moiety of PBG.
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