ACID STABILIZATION OF HUMAN GROWTH-HORMONE EQUILIBRIUM FOLDING INTERMEDIATES

Equilibrium denaturation experiments were performed on human growth ho rmone (hGH) under acidic conditions (pH 1.5-3.0) and different protein concentrations. At 0.1 mg/ml hGH using intrinsic tryptophan fluoresce nce and far-UV circular dichroism (CD) detection, midpoint values of 4 .6 M GdnHCl were observed that are identical to those obtained at neut ral pH. However, the Delta G values were reduced at pH 2.5 relative to pH 8.0 (10.5 vs. 15 kcal/mol). increasing the protein concentration t o 1 mg/ml resulted in a biphasic denaturation profile by far-UV CD det ection at 222 nm, while near-UV CD measurements at 295 nm yielded a co operative transition with a midpoint value of 3.6 M GdnHCl. These resu lts indicate that equilibrium intermediates having a propensity to agg regate are highly populated under acid conditions. Static light scatte ring measurements performed under partial unfolding conditions (4.5 M GdnHCl) at pH 2.5 confirmed the existence of a large molecular weight (similar or equal to 80 kDa) self-associated intermediate. No evidence of aggregation was found for hGH under acid conditions in the absence of denaturant, indicating that self-association results from the form ation of an intermediate. Equilibrium GdnHCl concentration-jump experi ments confirmed that association only occurs from an intermediate spec ies and not from any other conformational state, and formation of the self-associated intermediate can lead to irreversible loss of protein due to precipitation. These results demonstrate that acid stabilizes e quilibrium folding intermediates of hGH.