Studies On Aggregate And Intermediate Formed During The Dilution Refolding Procedure Of Egg White Lysozyme

In this paper, the mechanism of protein refolding is studied with egg white lysozyme as the model protein.The intermediate formed during the dilution refolding procedure of denatured egg white lysozyme was studied by "phase diagram" method of fluorescence. And the aggregate formed during the procedure of dilution refolding denatured-reduced and denatured egg white lysozyme was researched by the method of lauryl sodium sulfate-polyacrylamide gel electrophoreses (SDS-PAGE) and cathode polyacrylamide gel electrophoreses ( PAGE) and high-performance size-exclusion chromatography (SEC) .The refolding of unfolded egg white lysozyme which was induced by urea and guanidine hydroehloride, had been studied by "phase diagram" method of fluorescence. The results showed that there won'be any intermediate be formed during the refolding procedure when the reducer was present in the unfolding system, showing the dilution refolding procedure obeys a typical two-state model under such conditions;However two intermediates were formed during the refoding procedure when the reducer was absent in the unfolding system, indicating the dilution refolding procedure follows a four-state model under such conditions.When the denatured-reduced egg white lysozymes were diluted by the dilution refolding buffer solution, an observable amount of aggregate precipitates would immediately be formed. Results of non-reducing SDS-PAGE of supernatant and precipitate showed that besides being directly refolded to native lysozyme molecules, denatured-reduced lysozyme molecules could also form bi-molecular and tri-molecular lysozyme aggregates. The SEC result of supernatant also confirmed that the supernatant contained tri-molecular aggregate. Reducing SDS-PAGE results of supernatant and precipiate showed that bi-molecular and tri-molecular lysozyme aggregates were formed chiefly through wrongly linked intermolecular disulfide bonds between the denatured-reduced lysozyme molecules. The cathode PAGE of precipitate and the refolding results in the presence of SDS showed that bi-molecular aggregate was soluble and they can formed tetra-molecular aggregate precipitate through intermolecular non-covalent bonds interaction.The non-reducing SDS-PAGE of the refolding solution of denatured lysozyme showed that no aggregate formed during the refolding procedure when the concentration of lysozyme was below 7.0mg/mL;While bi-molecular andtri-molecular egg white lysozyme aggregate could be formed during the refolding procedure when the concentration of lysozyme was above 7.0 mg/mL. Cathode PAGE of refolding solution indicated that there was not any aggregate be accumulated during the refolding procedure when the concentration of lysozyme was below 4.0 mg/mL;While bi-molecular and tri-molecular lysozyme aggregates were formed during the refolding procedure, when the concentration of lysozyme was above 4.0 mg/mL. The SEC result of refolding solution also supported the results of protein electrophoreses.Based on the results of experiments ,we can draw the following conclusions. ( I ) The refolding procedure of denatured-reduced egg white lysozyme is a two-state procedure . The dilution refolding procedure of denatured-reduced egg white lysozyme in urea solution can be discribed as :during the refolding procedure,soluble bi-molecular and tri-molecular egg white aggregates were initially formed through intermolecular misconnection of disulfide bonds between egg white lysozyme molecules, then the soluble bi-molecular egg white lysozyme aggregates can further form tetra-molecular precipitates through the non-covalent interactions.(II) The dilution refolding procedure of denatured lysozyme according with a four-state procedure and two intermediates were formed during the refolding procedure.The refolding procedure of denatured lysozyme in urea solution can be described as : the refolding procedure does not form aggregate when the concentration of lysozyme was below 4.0 mg/mL;When the concentration of lysozyme was between 4.0 and 7.0 mg/mL,some intermediates can form bi-molecular and tri-molecular aggregates through non-covalent bonds interactions;While the concentration of lysozyme was above 7.0 mg/mL,some intermediates can form aggregate gel.