J. Biochem, 1976, Vol. 80, No. 2 229-237
© 1976 Japanese Biochemical Society
research-article |
The Chemical Modification of Beef Liver Catalase: V. Ethoxyformylation of Histidine and Tyrosine Residues of Catalase with Diethylpyrocarbonate
Division of Biochemistry, The University of Tsukuba School of Medicine Niihari-gun, Ibaraki 300–31
In order to elucidate the possible roles of histidine and tyrosine residues of catalase (EC 1.11.1. 6) in maintaining the quaternary structure and catalatic activity, diethyl-pyrocarbonate modification experiments were carried out.
A method for the estimation of N-ethoxyformyl (EF)-His at pH 5–7 and of O-ethoxyformyl (EF)-Tyr in alkaline solution by measuring A242 nm (
mM=3.2) and A278 nm (italicmM=1.16), respectively, was developed. The formation of EF-His and EF-Tyr was an electrophilic reaction and was dependent on pH, exhibiting pK values of 6.8 and 9.9, respectively. The maximal yield of EF-His at pH 6.0 was 49% of the total histidine content, but no inactivation nor unfolding of the enzyme was observed. The formation of 12 EF-Tyr residues per mole of catalase at pH 8.1 did not cause any inactivation, but the formation of 8 more EF-Tyr residues at pH 8.9 resulted in both inactivation and unfolding. Nearly complete inactivation and partial splitting of catalase were observed when 43–46 EF-Tyr residues per mole were produced at pH 10.0. More EF-His residues were formed by the reaction of diethyl pyrocarbonate with cyanoethylated (CE)-catalase monomer (subunit) than with CE-catalase tetramer. The CE-catalase tetramer and monomer were extensively O-ethoxyformylated, reaching 100% EF-Tyr formation. These results indicate that a half of the histidine residues may lie outside the protein core and that three-quarters of the tyrosine residues are probably in the protein core of the enzyme. The production of 2–3 EF-Tyr residues per mole of the monomer by ethoxyformylation at pH 7.0 was accompanied by a decrease in the magnitude of the Soret peak. A possible interaction of those tyrosine residues with porphyrin of the heme group is discussed.