Reconstitution of alkaline-denatured catalase

Bovine liver catalase at pH 12 dissociates into four physically indistinguishable sub-units and loses its enzymatic activity. The dissociation is accompanied by (1) changes in the hydrodynamic properties (its intrinsic viscosity increases from 0.039 to 0.23 dl/g and sedimentation coefficient decreases from 11.4 to 2.85 S), (2) the disappearance of the Soret band at 405 mμ characteristic of heme proteins and the appearance of a small broad peak at 420 mμ, (3) the disappearance of the Cotton effects in the Soret band, and (4) the reduction of the 233-mμ trough of the Cotton effects in the ultraviolet region to about one half in magnitude, suggesting the reduction of helical content. However, the reconstituted catalase molecule has the same physical and chemical properties as the native protein. The subunits can be reconstituted by rapidly diluting the alkaline solution with a pH 7 phosphate buffer. The percentage of enzymatic activity recovered depends on the time required for denaturation and renaturation, concentration of the protein, dilution volume, and temperature of the solution.