/ . Biochem., 78, 641-651 (1975)
Oxidation of Tryptophan in Lysozyme by Ozone in Aqueous Solution
Division of Protein Chemistry, Institute for Protein Research, Osaka University, Suita, Osaka 565 Received for publication, April 7, 1975
A tryptophan residue in hen's egg-white lysozyme [EC 3.2.1.17] was modified by ozone in an aqueous solution. One of the six tryptophan residues in the enzyme was oxidized to N'-formylkynurenine with concomitant loss of the enzymatic activity. Physicochemical studies of this modified enzyme (OL-I) revealed that the ozonization of lysozyme in aqueous media resulted in little change of the gross molecular conformation. It was deduced that the modified tryptophan residue in OL-I was possibly located in position 62 (or 63) of the protein.
bottom of a cleft capable of interacting with the f-carboxyl group of glutamic acid 35, which forms a part of the catalytic site (7). Since the conformation of the native protein was thought to be altered significantly in anhydrous formic acid, we have studied the ozonization of lysozyme in aqueous media in order to preserve the native conformation of the enzyme during the oxidative modification reaction. It was found that, at the stage of oxidation of one out of six tryptophan residues to N'-formylkynurenine, the lytic activity of lysozyme decreased dramatically. In the present report we describe some of physicochemical properties of the inactivated enzyme which had lost one tryptophan by this oxidation (OL-I) and the results of analysis of the peptide containing N'-formylkynurenine. These results suggest that the tryptophan residue oxidized was located at position 62 (or 63), leading to inactivation without altering the main chain conformation.
Oxidative cleavage of a double bond in organic compounds by ozone has generally been carried out in both protic and aprotic solvents (J). Among these solvents, formic acid has been utilized to oxidize the indole nucleus of tryptophan in proteins to correlate sensitive tryptophan residues with the biological activity of proteins (2, 3). Previero et al. reported that the oxidation of tryptophan in positions 108 and 111 of hen's egg-white lysozyme [EC 3. 2.1.17] to N'-formylkynurenine in anhydrous formic acid produced no inactivation of its lytic activity towards the cell wall of Micrococcus lysodeikticus (4). However, oxidation of tryptophan in lysozyme in aqueous solutions with different oxidants such as N-bromosuccinimide (5) and iodine (
Oxidation of Tryptophan in Lysozyme by Ozone in Aqueous Solution
Division of Protein Chemistry, Institute for Protein Research, Osaka University, Suita, Osaka 565 Received for publication, April 7, 1975
A tryptophan residue in hen's egg-white lysozyme [EC 3.2.1.17] was modified by ozone in an aqueous solution. One of the six tryptophan residues in the enzyme was oxidized to N'-formylkynurenine with concomitant loss of the enzymatic activity. Physicochemical studies of this modified enzyme (OL-I) revealed that the ozonization of lysozyme in aqueous media resulted in little change of the gross molecular conformation. It was deduced that the modified tryptophan residue in OL-I was possibly located in position 62 (or 63) of the protein.
bottom of a cleft capable of interacting with the f-carboxyl group of glutamic acid 35, which forms a part of the catalytic site (7). Since the conformation of the native protein was thought to be altered significantly in anhydrous formic acid, we have studied the ozonization of lysozyme in aqueous media in order to preserve the native conformation of the enzyme during the oxidative modification reaction. It was found that, at the stage of oxidation of one out of six tryptophan residues to N'-formylkynurenine, the lytic activity of lysozyme decreased dramatically. In the present report we describe some of physicochemical properties of the inactivated enzyme which had lost one tryptophan by this oxidation (OL-I) and the results of analysis of the peptide containing N'-formylkynurenine. These results suggest that the tryptophan residue oxidized was located at position 62 (or 63), leading to inactivation without altering the main chain conformation.
Oxidative cleavage of a double bond in organic compounds by ozone has generally been carried out in both protic and aprotic solvents (J). Among these solvents, formic acid has been utilized to oxidize the indole nucleus of tryptophan in proteins to correlate sensitive tryptophan residues with the biological activity of proteins (2, 3). Previero et al. reported that the oxidation of tryptophan in positions 108 and 111 of hen's egg-white lysozyme [EC 3. 2.1.17] to N'-formylkynurenine in anhydrous formic acid produced no inactivation of its lytic activity towards the cell wall of Micrococcus lysodeikticus (4). However, oxidation of tryptophan in lysozyme in aqueous solutions with different oxidants such as N-bromosuccinimide (5) and iodine (
