EQUILIBRIUM DENATURATION OF RECOMBINANT MURINE INTERLEUKIN-6 - EFFECT OF PH, DENATURANTS, AND SALT ON FORMATION OF FOLDING INTERMEDIATES
- Author(s)
- Ward, LD; Matthews, JM; Zhang, JG; Simpson, RJ;
- Details
- Publication Year 1995-09-19,Volume 34,Issue #37,Page 11652-11659
- Journal Title
- BIOCHEMISTRY
- Publication Type
- Journal Article
- Abstract
- The equilibrium denaturation of an Escherichia coli-derived recombinant murine interleukin-6 (mIL-6) was studied using fluorescence and circular dichroism spectroscopy. The urea-induced unfolding of mIL-6 at pH 4.0 can be described by a two-state unfolding mechanism based on the superimposibility of the CD and fluorescence unfolding transitions. Assuming a two-state mechanism and a linear dependence of the free energy of unfolding on denaturant concentration, a value of 6.9-9.0 kcal/mol was calculated for the free energy of unfolding in the absence of denaturant [Delta G(U)(H2O)]. However, when GuHCl was used as a denaturant at pH 4.0, a biphasic unfolding transition was observed. This unfolding transition has a distinct midpoint occurring at 2.5 M GuHCl, which is indicative of the formation of stable folding intermediates. Similar intermediate folded species were also observed at pH 7.4 when either urea or GuHCl were used as denaturants. The intermediate folded states of mIL-6 exhibited a tendency to aggregate, as judged by the concentration dependence of their fluorescence characteristics. The fluorescence emission maximum of mIL-6 at pH 7.4 in the presence of 1.5 M GuHCl, for example, was blue-shifted from 343 nm at a protein concentration of 50 mu g/mL to 336 nm at 500 mu g/mL. Intermediate formation at pH 4.0, using 10 mM sodium acetate buffer and urea as the denaturant, was facilitated by the addition of 0.4 and 0.8 M salt, where the salt was either NaCl or GuHCl. These data, together with the pH-dependent fluorescence characteristics, suggest that ionic effects and the charged state of mIL-6-particularly in the region of Trp36-play an important role in the formation of folding intermediates and aggregation.
- Publisher
- AMER CHEMICAL SOC
- Keywords
- BOVINE GROWTH-HORMONE; MOLTEN GLOBULE STATE; AMINO-ACID SEQUENCE; CONFORMATIONAL STABILITY; GUANIDINE-HYDROCHLORIDE; STIMULATING FACTOR; PROTEIN; PURIFICATION; CELLS; PROLIFERATION
- Publisher's Version
- https://doi.org/10.1021/bi00037a002
- Terms of Use/Rights Notice
- Refer to copyright notice on published article.
Creation Date: 1995-09-19 12:00:00