DESIGN OF OPTIMUM REFOLDING SOLUTION BY COMBINATION OF REAGENTS CLASSIFIED BY SPECIFIC FUNCTION

The effect of guanidinium chloride on protein unfolding was regarded a s a combination of the effects on interactions such hydrophobic intera ction or hydrogen bond, and ionic interaction that construct a higher- order structure of the protein. Urea and LiCl instead of guanidinium c hloride were used for refolding of denatured reduced lysozyme by assum ing that urea and LiCl individually control hydrophobic interaction or hydrogen bond and ionic interaction, respectively. The refolding medi a were prepared using various combinations of urea and LiCl concentrat ions. Reoxidation of thiol groups was carried out in a glutathione red ox system. The recovered enzyme activity at a lysozyme concentration o f 10 mu M was usually less than 20% in 50 mM Tris buffer due to irreve rsible misfolding and/or aggregation during refolding. In the presence of urea and LiCl at high concentrations the recovered activity increa sed markedly. Complete renaturation could be achieved in a broad range of concentrations of urea and LiCl around the optimum of 3 M urea and 1.5 M LiCl, while it could not be achieved solely with guanidinium ch loride. The availability of the concept that multifunction of guanidin ium chloride can be separated approximately into two monofunctions of urea and LiCl was confirmed by the optimum design of refolding media p repared by combinations of urea and LiCl. A characteristic diagram for the refolding of lysozyme was depicted. This diagram will aid the opt imum design of refolding media and study of the refolding mechanism.