In Vitro Assembly of l-Asparaginase Subunits

The reassociation of denatured subunits of l-asparaginase from Escherichia coli B was examined after dilution into buffer and into dilute urea solution. By gradually increasing the urea content of the reassociation mixture, we have attempted to elucidate the steps through which the denatured polypeptide reassembles into the catalytically active tetramer. Refolding of the chain is complete within 30 s after dilution of the denaturant and this is followed by a slower aggregation of the subunits into the oligomers. There was no evidence of dimers formed as intermediates in the reassembly process. The lone tryptophan which is present in the subunit is transferred from an aqueous to a hydrophobic environment during the refolding process while many of the tyrosyl residues undergo similar changes in their milieu as the subunits aggregate. l- and d-Asparagine are equally effective in inducing association of monomer to tetramer in the presence of urea and under assay conditions this process requires approximately 5 min. Both stereoisomers of aspartic acid are also partially effective in promoting aggregation; however, l-glutamine and glycyl-l-asparagine are rather ineffective as promoters of the assembly process.