Protein phase diagrams II: nonideal behavior of biochemical reactions in the presence of osmolytes

Biophys J. 2007 Jan 1;92(1):245-56. doi: 10.1529/biophysj.106.092262. Epub 2006 Oct 6.


In the age of biochemical systems biology, proteomics, and high throughput methods, the thermodynamic quantification of cytoplasmatic reaction networks comes into reach of the current generation of scientists. What is needed to efficiently extract the relevant information from the raw data is a robust tool for evaluating the number and stoichiometry of all observed reactions while providing a good estimate of the thermodynamic parameters that determine the molecular behavior. The recently developed phase-diagram method, strictly speaking a graphical representation of linkage or Maxwell Relations, offers such capabilities. Here, we extend the phase diagram method to nonideal conditions. For the sake of simplicity, we choose as an example a reaction system involving the protein RNase A, its inhibitor CMP, the osmolyte urea, and water. We investigate this system as a function of the concentrations of inhibitor and osmolyte at different temperatures ranging from 280 K to 340 K. The most interesting finding is that the protein-inhibitor binding equilibrium depends strongly on the urea concentration--by orders-of-magnitude more than expected from urea-protein interaction alone. Moreover, the m-value of ligand binding is strongly concentration-dependent, which is highly unusual. It is concluded that the interaction between small molecules like urea and CMP can significantly contribute to cytoplasmic nonideality. Such a finding is highly significant because of its impact on renal tissue where high concentrations of cosolutes occur regularly.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Biophysics / instrumentation
  • Biophysics / methods*
  • Cytoplasm / metabolism
  • Dose-Response Relationship, Drug
  • Kidney / metabolism
  • Kinetics
  • Ligands
  • Protein Binding
  • Protein Denaturation
  • Proteins / chemistry*
  • Ribonuclease, Pancreatic / chemistry
  • Temperature
  • Thermodynamics
  • Urea / chemistry
  • Water / chemistry


  • Ligands
  • Proteins
  • Water
  • Urea
  • Ribonuclease, Pancreatic