The Effect Of Refolding And Structure Of Lysozyme Assisted By Small Molecular Additives

In order to analyze the mechanism of small molecular additives assisting protein refolding and to develop new valued protein refolding assistant, the character and differences of various dilution additives on the refolding of denatured-reduced chicken egg white lysozyme were investigated, using Fluorescence Spectroscopy and Circular Dichroism to analyze. Based on the protection of trehalose and betaine to protein, the refolding effect of trehalose and betaine on lysozyme under different GdnCl concentration and lysoyzme concentration was researched, as well as the kinetics was examined.It was founded that the renaturation yield was improved in the presence of arginine, thiourea, acetone, acetamide and glycerol compared with no additives. The role of arginine to assist protein refolding was similar to that of acetone, both of them were able to combine with the denature lysozyme and improve the refolding yield. And the content ofαhelix of refolding and nature lysozyme was increased with the appearance of arginine and acetone, and both of them had the resemble function on the structure of lysozyme. Acetamide and thiouera had the parallel effect with GdnCl on the renaturation of lysozyme , both of them were able to prevent the aggregation of peptide, and at the high concentration they were able to restrain the refolding of peptide , but the structure of lysozyme under experiment condition could not be destroyed by them. The effect of glycerol was to enhance the viscosity of molecular environment of lysozyme by increasing the concentration to assist recovery of lysozyme and to keep the high content ofαhelix of lysozyme .At the low GdnCl concentration , the content ofαhelix was substantive with high concentration of trehalose ; at 2 mol/L GdnCl , the content ofαhelix andβsheet was decreased because of the cooperation of trehalose with GdnCl which led to the loss of activity of lylsozyme. Whether refolding denature lysozyme at low GdnCl concentration or protecting lysozyme at high GdnCl concentration, the function of betaine was to retain the structure of lysozyme so as to increase the recover yield of refolding lysozyme and keep the activity of nature lysozyme. Both of trehalose and betaine could prevent aggregation of peptide , but the high concentration of trehalose enhanced the content ofαhelix to decrease the recover yield; however betaine could maintain the structure of lysozyme to keep the recover yield at a high level.