title: Evaluation of the physical stability of the EC5 domain of E-cadherin: effects of pH, temperature, ionic strength, and disulfide bonds.
abstract: The development of protein drugs has been hampered by difficulties in formulating them due to their inherent chemical and physical stability, which could generate problems during the late stages of development. Thus, a basic understanding of the effect of structural features on the physicochemical stability of proteins can provide fundamental solutions to the formation of proteins. In this work, the physical stability of the EC5 protein under variable pH, temperature, and ionic strength and the role of the disulfide bond on the physical stability of EC5 were evaluated. All spectroscopic measurements were integrated in empirical phase diagrams, and these diagrams showed the stable and unstable regions of EC5. The native EC5 is more stable at high than at low ionic strength in a wide pH range during temperature elevation to 70 degrees C. The empirical phase diagrams also show that the reduced EC5 has lower stability than the parent EC5. The reduced EC5 has secondary structure only at pH 3 and 4 and is unfolded at other pH values. Finally, the reduced EC5 rapidly forms a precipitate at pH 4 and 5 upon heating. In conclusion, this study shows that ionic strength and the presence of the disulfide bonds are critical for the stability of EC5.
authors.affiliation: Department of Pharmaceutical Chemistry, The University of Kansas, 2095 Constant Ave., Lawrence, Kansas 66047, USA.
authors.forename: Kai
authors.initials: K
authors.lastname: Zheng
authors.forename: C Russell
authors.initials: CR
authors.lastname: Middaugh
authors.forename: Teruna J
authors.initials: TJ
authors.lastname: Siahaan
mesh_terms: D015820:Cadherins / Q000737:chemistry*; D004220:Disulfides / Q000737:chemistry*; D004353:Drug Evaluation, Preclinical / Q000379:methods; D004355:Drug Stability; D006860:Hydrogen Bonding; D006863:Hydrogen-Ion Concentration; D009994:Osmolar Concentration; D017434:Protein Structure, Tertiary; D013696:Temperature
chemical_list: D015820:Cadherins; D004220:Disulfides