Photochemical Inactivation of Lactoperoxidase Sensitized by Carbonyl Compounds

Abstract

1. 2,3-Butanedione was found to be a poor noncompetitive inhibitor (Ki z 0.1 M) of bovine milk lactoperoxidase. Irradiation of an incubation mixture of these two substances in the visible region led, however, to an irreversible loss of enzyme activity. The inactivation rates were dependent on light intensity, light wavelength and sensitizer concentration. Maximal rate of inactivation occurred at 400 -420 nm. Other dicarbonyl compounds which caused a similar photodestruction of lactoperoxidase in the visible region were 2,3-pentanedione and 1 -phenyl-I ,2-propanedione, the following compounds being without effect : phenylglyoxal, methylglyoxal, glyoxal, 1,2-cyclohexanedione and acetone. Of the latter, however, 1,2-cyclohexanedione, phenylglyoxal, methylglyoxal and acetone inactivated lactoperoxidase at 254 nm.2. The butanedione-sensitized photochemical inactivation of lactoperoxidase was preventcd by the presence of free radical scavengers and singlet oxygen quenchers, the best protectors being the histidine derivatives ~-3-methylhistidine, L-I-methylhistidine and i.-thiol-2-histidine, which displayed dual quenching ability. No photochemical inactivation of lactoperoxidase occurred in anoxic media even in the presence of high butanedione concentration and strong illumination.3. The butanedione-sensitized photochemical inactivation of lactoperoxidase was accompanied by extensive changes in the ultraviolet absorption spectra of the enzyme. Simultaneously, a dramatic loss in tryptophan fluorescence occurred. All spectral and fluorescence changes were prevented by the above protectors. The sensitized enzyme inactivation was shown to be an all-or-none type of reaction ; no partially inactivatcd enzymes existed.4. Amino acid analysis of native and butanedione light-treated lactoperoxidase showed that the treated enzyme contained considerably less of the following amino acids than the native enzyme : methionine, arginine, tyrosine, aspartic acid, serine, glutamic acid. Histidine residues were probably attacked also. Inhibition and modification studies showed that the activity of the enzyme was not based on arginyl and tryptophyl residues.5. These results suggest that 2,3-butanedione, 1,2-cyclohexanedione, phenylglyoxal and related dicarbonyl compounds, which have previously been used as relatively specific probes for the active arginyl residues of enzymes, cause under suitable modification conditions extensive changes in the structure of an enzyme by affecting a multitude of amino acid residues. These compounds can totally inactivate an enzyme regardless of the essentiality or inessentiality of arginyl residues for enzyme activity. The present results warn against drawing conclusions from modification studies with dicarbonyl Compounds unless the irradiation conditions are carefully considered.2,3-Butanedione has in numerous studies been reported to demonstrate high selectively toward active arginyl residues of enzymes [l -141. A typical case is the modification of an active arginyl residue of carboxypeptidase A with thi...

Affinity Labeling of Arginyl Residues at the Catalytic Region of Estradiol 17β‐Dehydrogenase from Human Placenta by 16‐Oxoestrone

“…Estradioll7~-dehydrogenase of human placenta was reported to retain 22 residues of arginine per subunits buffer at pH > 8 in the dark [15]. However, a-chymotrypsin [16] and pepsin [17] in the absence of the borate ion, and aminopeptidase [18] and lactoperoxidase [19] in the borate buffer at neutral pH were inactivated by butanedione as a photosensitizing agent. The inactivation rate of the estradiol 17P-dehydrogenase by 16-oxoestrone under the laboratory illumination or ultraviolet light at 254 nm was the same as that in the dark.…”

On the water-promoted mechanism of peptide cleavage by carboxypeptidase A. A theoretical study

“…CPA) is a zinc metalloexopeptidase of 307 residues (MW = 34 472), and a well-studied enzyme (1)(2)(3)(4)(5)(6). This protease preferentially hydrolyzes peptide or ester bonds at the C-terminus of polypeptide substrates with hydrophobic (aromatic or aliphatic) residues.…”

Photoaffinity modification of .DELTA.5-3-ketosteroid isomerase by light-activatable steroid ketones covalently coupled to agarose beads

“…Chromophore-sensitized photooxidation involves the intermediacy of singlet oxygen, and ketones have been shown to be effective sensitizers of photooxidation reactions of proteins (Mákinen & Mákinen, 1982a). Histidine and several other imidazole-containing compounds have been shown to be efficient quenchers of ketone-sensitized photooxidation (Mákinen & Mákinen, 1982b) of lactoperoxidase. As shown in Table IV,2.5 mM and larger concentrations of L-histidine were found to suppress the oxygen-stimulated nonspecific inactivation processes and to result in full protection by sodium cholate under aerobic conditions.…”

“…One possibility for an oxygen-dependent photochemical reaction is chromophore-(dye) sensitized photooxidation (Means & Feeney, 1971). Mákinen & Mákinen (1982b) have recently made a survey of inhibitors of chromophore-sensitized photooxidation, and these workers have found that various histidine-type compounds are very efficient scavengers of the singlet oxygen that is believed to be the active oxidant in these reactions. In order to assess the importance of singlet oxygen in the aerobic inactivations, a study of the effect of L-histidine of the extent of inactivation and the protective effects of sodium cholate was made.…”