title: Kinetic and thermodynamic analysis of thermal unfolding of recombinant erythropoietin.
abstract: Thermal stress was used to assess the stability of recombinant human erythropoietin (EPO) derived from Chinese hamster ovary cells. In 20 mm phosphate at pH 7.0, this protein had a highly reversible thermal unfolding as observed by far UV circular dichroism (CD) and native gel analysis, with no indication of protein aggregation. It had a relatively low melting temperature at 53 degrees C. Assuming a two-state transition, the observed reversibility permits thermodynamic analysis of the unfolding of EPO, which shows that the free energy of unfolding at 25 degrees C is only 6-7 kcal/mol. Upon heating to 79 degrees C over 30 min, however, this protein does undergo aggregation as assessed by native gel. In 20 mm phosphate and citrate at pH 7.0, the results are similar, i.e., EPO suffered a substantial aggregation, while it showed little aggregation in 20 mm Tris or histidine at pH 7.0 and 20 mm glycine at pH 6.3 under identical heat treatment.
authors.affiliation: Alliance Protein Laboratories, Thousand Oaks, CA 91360, USA. tarakawa2@aol.com
authors.forename: T
authors.initials: T
authors.lastname: Arakawa
authors.forename: J S
authors.initials: JS
authors.lastname: Philo
authors.forename: Y
authors.initials: Y
authors.lastname: Kita
mesh_terms: D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D004921:Erythropoietin / Q000737:chemistry*; D007700:Kinetics; D011487:Protein Conformation; D017510:Protein Folding; D011994:Recombinant Proteins; D012997:Solvents; D013056:Spectrophotometry, Ultraviolet; D013696:Temperature; D013816:Thermodynamics; D014461:Ultracentrifugation
chemical_list: D011994:Recombinant Proteins; D012997:Solvents; D004921:Erythropoietin