Folding Units of Reduced and S-Protected Lysozyme

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At the present, it in accepted that protein folding proceeds through early folding of short segments of a polypeptide chain and the subsequent assembly -of them into the final tertiary structure. Lysozyme is a protein containing four disulfide bridges, and its tertiary structure in very stable in the aqueous solution. When it loses the disulfide bridges, however, its tertiary structure can not be kept. In such an unfolded state of lysozyme, local structures are expected to be prefferentailly formed through short range interactions within shoot segments of polypeptide chain. It is important for the study of protein folding to find such preferential folding units and to identify their positions along a polypeptide chain.Reduced and S-3-(trimethylated amino) propylated lysozyme (abbreviated to TMAP-lysozyme) was used for our experiments. Circular dichroism spectra off TMAP-lysozyme were measured in various solutions containing GuHC1 or sorbitor. The addition of sorbitor was found to increase the negative ellipticity in the 190-260 nm region. The limited proteolysis of this TMAP-lysozyme by trypsin was performed with the aim of identifying the segments of polypeptide chain havens the preferential local structures. All the tryptic peptides of TNAP-lyscyzyme were identified on the reverse phase chromaturams. The time course of production of their tryptic peptides was followed by plotting their peak areas against the reaction time with trypsin. Also, intermediate products consisting of several tryptic peptides were found on the same chromatgrams. Kinetics of their production and digestion was also observed. Hydrolysis rate constants by trypsin were estimated at all cleavage sites according to the Michaelis Menten mechanism. As a result, it was found that the sites of resistance to trypsin concentrate in the N-terminal half of the polypeptide chain of TMAP-lysozyme.