Dissociation of beef liver catalase in its subunits

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Earlier results from other laboratories have shown that beef liver catalase is built up of subunits. However there was no integral relationship between the number of subunits and the number of active sites (heme groups). To clarify the question how the heme groups are distributed among the subunits, the quaternary structure of catalase was studied under different denaturing conditions. 1 5 M guanidine-HCl, in the presence of 0.1 M β-mercaptoethanol, caused dissociation of catalase into subunits each with a molecular weight of 59,000 (s°20, w= 2.10 S; D°20, w= 3.20 F). In the absence of β-mercaptoethanol the molecular weight in 5 M guanidine-HCl was found to be about 140,000 (s°20, w= 3.50; D°20, w= 2.17 F). 2 Succinylation of native catalase with succinic anhydride at neutral pH yielded subunits each with a molecular weight of 65,000 (s°20, w= 3.22 S; D°20, w= 4.43 F). 3 After short incubation (up to six hours) at pH 12.5–12.8 catalase dissociated into subunits each with a sedimentation coefficient (s°20, w) of 2.89 S. The molecular weight of these subunits was estimated to be 54,000 and 67,000 respectively. The first value was obtained by combining sedimentation and viscosity measurements ([η] = 17.78 ml/g) from the relation deduced by Scheraga and Mandelkern using a β-value of 2.5 × 106. The principle of Archibald was used for the estimation of the second value. Longer incubation (e.g. twenty hours) caused splitting into several components, the main component after sixty hours possesses a sedimentation coefficient of 1.7 S. Probably this splitting is due to hydrolysis of peptide bonds. For this reason it was not possible to estimate the diffusion coefficient of the S2.9-component which needs a dialyzed solution. 4 During the incubation of native catalase in the presence of 7.3–14.6 mM sodium dodecyl sulfate an incomplete dissociation into a component with s°20, w= 3.4 S was observed. Separation of the dissociation product by means of a separation cell yielded a single component with a molecular weight of about 80,000 (s°20, w= 3.3 S; D°20, w= 3.3 F). The significance of this component is not clear, because the molecular weight disagrees with the value of 61,000 obtained by all other methods. Treatment of catalase or apocatalase with formic acid followed by incubation with sodium dodecyl sulfate caused dissociation into subunits with a sedimentation coefficient of 3.8 S (D°20, w= 5.1 F) and 4 S respectively. The molecular weight of the S3.8-component was found to be 60,000. 5 8 M urea resulted in a dissociation into subunits each with a molecular weight of 133,000 (s°20, w= 3.3 S; D°20, w= 2.23 F). About the same molecular weight (120,000) was obtained by Samejima and Yang at pH 3. By comparison with the molecular weight of the native enzyme (240,000–250,000) it was concluded that beef liver catalase consists of four subunits of equal size (molecular weight 60,000–65,000). This conclusion was confirmed by electron microscopic studies. In 5 M guanidine-HCl, or in 8 M urea, or at pH 3 the catalase molecule dissociated into two subunits only, whereas all other denaturing conditions used gave rise to four subunits. Together with the reported renaturation experiments the dissociation scheme of beef liver catalase is shown in the following equation: As mentioned above guanidine-HCl (and probably urea), in the presence of β-mercaptoethanol, caused dissociation into four subunits, ẃhereas in the absence of β-mercaptoethanol only two subunits were obtained. It is assumed that the reason for this difference might be the oxidation of SH groups to disulfide bridges during the denaturation linking subunits together. This process is prevented in the presence of β-mercaptoethanol. In the native enzyme molecule the four subunits are held together only by noncovalent bonds. It was concluded that each of the four subunits contains one active center.