On the denaturation of porcine erythrocyte catalase with alkali, urea, and guanidine hydrochloride in relation to its subunit structure.
The dissociation of porcine erythrocyte catalase [EC 1.11.1.6] into subunits on denaturation with alkali, GuHCl and urea was investigated by following the changes in hydrodynamic properties, absorption and CD spectra in the Soret region and inactivation of the enzyme. It was found that dissociation proceeded in an "all or none" manner from the native tetramer (molecular weight, ca. 250,000) into identical 1/4-sized monomers (molecular weight, ca. 54,000 with alkali, 65,000 with urea and 71,000 with GuHCl) as estimated by ultracentrifugal analyses. On this dissociation, the sedimentation coefficient decreased from about 11S to 5.1 - 3.7S, and absorption spectra in the Soret region decreased to about 40% of the native level and showed a broad band around 365-375 nm and a shoulder around 415-420 nm; these changes were accompanied by complete loss of enzyme activity. The change in enzyme activity correlated well with that of absorption and CD spectra in the Soret region, depending on denaturation time, alkaline pH used and concentration of both denaturants. The reassociated catalase obtained by removing urea by dialysis was characterized by recovery of distinct CD bands in the Soret and near ultraviolet regions, although the partial refolding of alpha-helical conformation occurred without recovery of enzyme activity. These results indicate that the conformational changes and dissociation process of catalase into subunits can be monitored spectrophotometrically in relation to enzyme activity, and that subtle conformations near the heme groups and polypeptide backbone play an important role in maintaining full enzyme activity of the catalase molecule.
D000468:Alkalies / Q000494:pharmacology; D000818:Animals; D002374:Catalase / Q000097:blood*; D055598:Chemical Phenomena; D002621:Chemistry; D002942:Circular Dichroism; D004912:Erythrocytes / Q000201:enzymology*; D019791:Guanidine; D006146:Guanidines / Q000494:pharmacology; D066298:In Vitro Techniques; D010446:Peptide Fragments / Q000032:analysis; D011487:Protein Conformation; D011489:Protein Denaturation; D013050:Spectrometry, Fluorescence; D013552:Swine; D014508:Urea / Q000494:pharmacology
D000468:Alkalies; D006146:Guanidines; D010446:Peptide Fragments; D014508:Urea; D002374:Catalase; D019791:Guanidine