DISSOCIATION, UNFOLDING AND REFOLDING TRIALS OF PIG-KIDNEY 3,4-DIHYDROXYPHENYLALANINE (DOPA) DECARBOXYLASE

The effect of guanidinium chloride (GuCl) on enzyme activity, hydrodynamic volume, circular dichroism. and fluorescence of 3,4-dihydroxyphenylalanine (Dopa) decarboxylase from pig kidney (pkDDC) was studied under equilibrium conditions. Unfolding proceeds in at least three stages. The first transition. occurring between 0 and 1 M GuCl. gives rise to a dimeric inactive species which has lost pyridoxal 5'-phosphate (PLP). and has a high tendency to aggregate, but retains almost all of the native spectroscopic characteristics. The second equilibrium transition, between 1 and 2.2 M GuCl. involves dimer dissociation, with some loss of tertiary and secondary structure. Additionally, gross conformational changes at or near the PLP microenvironment were detected by fluorescence of NaBH4-reduced enzyme. The third step, presumably representing complete unfolding of pkDDC. appears to be complete at 4.5 M GuCl, as indicated by the lack of further-substantial changes in any of the signals being studied. Attempts at refolding resulted in the findings that (1) partial reactivation is observed only starting from enzyme denatured at concentrations below 1.5 M GuCl. and (2) starting from completely denatured protein. the refolding process is apparently reversible down to concentrations of approx. (2) M GuCl. Taken together, this would seem to indicate that the monomer-dimer transition is impaired under the experimental conditions tested. A plausible model is presented for the unfolding/refolding of pkDDC.