Abstract
Equilibrium denaturation experiments were performed on human growth hormone (hGH) under acidic conditions (pH 1.5-3.0) and different protein concentrations. At 0.1 mg/ml hGH using intrinsic tryptophan fluorescence and far-UV circular dichroism (CD) detection, midpoint values of 4.6 M GdnHCl were observed that are identical to those obtained at neutral pH. However, the ΔG values were reduced at pH 2.5 relative to pH 8.0 (10.5 vs. 15 kcal/mol). Increasing the protein concentration to 1 mg/ml resulted in a biphasic denaturation profile by far-UV CD detection at 222 nm, while near-UV CD measurements at 295 nm yielded a cooperative transition with a midpoint value of 3.6 M GdnHCl. These results indicate that equilibrium intermediates having a propensity to aggregate are highly populated under acid conditions. Static light scattering measurements performed under partial unfolding conditions (4.5 M GdnHCl) at pH 2.5 confirmed the existence of a large molecular weight (≌ 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 formation of an intermediate. Equilibrium GdnHCl concentration-jump experiments confirmed that association only occurs from an intermediate species 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 equilibrium folding intermediates of hGH.
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
More From: Biochimica et Biophysica Acta (BBA)/Protein Structure and Molecular Enzymology
Disclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.