Preferential exclusion of sucrose from recombinant interleukin-1 receptor antagonist: role in restricted conformational mobility and compaction of native state

Proc Natl Acad Sci U S A. 1997 Oct 28;94(22):11917-22. doi: 10.1073/pnas.94.22.11917.

Abstract

Understanding the mechanism for sucrose-induced protein stabilization is important in many diverse fields, ranging from biochemistry and environmental physiology to pharmaceutical science. Timasheff and Lee [Lee, J. C. & Timasheff, S. N. (1981) J. Biol. Chem. 256, 7193-7201] have established that thermodynamic stabilization of proteins by sucrose is due to preferential exclusion of the sugar from the protein's surface, which increases protein chemical potential. The current study measures the preferential exclusion of 1 M sucrose from a protein drug, recombinant interleukin 1 receptor antagonist (rhIL-1ra). It is proposed that the degree of preferential exclusion and increase in chemical potential are directly proportional to the protein surface area and that, hence, the system will favor the protein state with the smallest surface area. This mechanism explains the observed sucrose-induced restriction of rhIL-1ra conformational fluctuations, which were studied by hydrogen-deuterium exchange and cysteine reactivity measurements. Furthermore, infrared spectroscopy of rhlL-1ra suggested that a more ordered native conformation is induced by sucrose. Electron paramagnetic resonance spectroscopy demonstrated that in the presence of sucrose, spin-labeled cysteine 116 becomes more buried in the protein's interior and that the hydrodynamic diameter of the protein is reduced. The preferential exclusion of sucrose from the protein and the resulting shift in the equilibrium between protein states toward the most compact conformation account for sucrose-induced effects on rhIL-1ra.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Chemical Phenomena
  • Chemistry, Physical
  • Circular Dichroism
  • Cysteine / chemistry
  • Deuterium Oxide
  • Electron Spin Resonance Spectroscopy
  • Humans
  • Interleukin 1 Receptor Antagonist Protein
  • Light
  • Models, Chemical
  • Protein Conformation / drug effects
  • Receptors, Interleukin-1 / antagonists & inhibitors*
  • Recombinant Proteins / chemistry
  • Scattering, Radiation
  • Sialoglycoproteins / chemistry*
  • Sialoglycoproteins / drug effects*
  • Spectrophotometry, Infrared
  • Spectrophotometry, Ultraviolet
  • Sucrose / pharmacology*
  • Surface Properties
  • Thermodynamics

Substances

  • IL1RN protein, human
  • Interleukin 1 Receptor Antagonist Protein
  • Receptors, Interleukin-1
  • Recombinant Proteins
  • Sialoglycoproteins
  • Sucrose
  • Deuterium Oxide
  • Cysteine