Dissociation of enzyme subunits in R141A enforced by imidazole. R141A as isolated is a mixture consisting of the native dimer and a small amount of a tetrameric form that is also active. The N-terminal His-tag causes the tetramerization . The absorbance traces are in arbitrary units (a.u.). Elution profiles are shown for R141A prior to (dashed line) and after incubation in the presence of 0.4 M imidazole for 30 min (dotted line), 60 min (solid line) and 140 min (dashed-dotted line). The observed peaks correspond to tetrameric (t; 362 kDa), dimeric (d; 181 kDa) and monomeric (m; 90.6 kDa) forms of the protein. A high-molecular mass peak is also visible in some traces, presumably showing soluble aggregated protein. Loss of phosphorylase activity in the presence of imidazole is correlated with the extent to which monomer formation had occurred (data not shown). Note that all samples contained the same protein concentration (0.4 mg/ml) prior to the incubation with imidazole. The decrease in peak area for the eluted protein forms as the incubation time in the presence of imidazole increased probably reflects loss of protein due to aggregation. Insoluble aggregates are removed by centrifugation prior to gel filtration. The protein concentration of the sample applied to the Superdex column was not measured.