Inhibition Of Covalent Binding Research Articles

The inhibition of covalent binding of the nascent complement component C4b to its target

The inhibition of covalent binding of the nascent C4b fragment of the human complement component to its natural target, immunoglobulin G, was studied. To this end, an immunoenzyme system was developed. In this ELISA method, the complement was activated on the sorbed IgG molecules and the resulting nascent C4b fragment acylated IgG or interacted with a competitive inhibitor added to the system. The inhibition constants for binding of the nascent C4b to its target were determined for immunoglobulins G1, G2, G3, G4, M, and A1, as well as for ferritin, yeast mannan, capsid polysaccharides of theNeisseria meningitidis A, B, and C serotypes, diphtheria anatoxin, epinephrine, and salicylic acid. On the basis of the experimental data, the immunoglobulin role at the activation stage of the complement regulation cascade, the relationship between the antigen immunogenicity and its ability to interact with C4b, and the direct effect of a number of therapeutic agents on the complement system were discussed. Lectins of various specificities were shown to inhibit the enzymic activation of C4 by the first complement component and the subsequent C4b sorption by its target, which allowed us to suggest that some oligosaccharide fragments of the C1s and C4 molecules are spatially close to the C1s active site and to the thioester bond of C4.

The microsomal metabolism of hexachlorobenzene: Origin of the covalent binding to protein

The musomal metabolism of hexachlorobenzene is studied, with special attention to the covalent binding to protein. The metabolites formed are pentachlorophenol and tetrachlorohy-droquinone. In addition, a considerable amount of covalent binding to protein is detected (250 pmoles pentachlorophenol, 17 pmoles tetrachlorohydroquinone and 11 pmoles covalent binding in an incubation containing 50 μmoles of hexachlorobenzene). In order to establish the potential role of reductive dechlorination in the covalent binding, the anaerobic metabolism of hexachlorobenzene was investigated. At low oxygen concentrations no pentachlorobenzene was detected, and only very small amounts of pentachlorophenol as well as covalent binding, indicating a relationship between covalent binding and the musomal oxidation of hexachlorobenzene. Incubations with 14C-pentachlorophenol at low concentrations showed that a conversion-dependent covalent binding occurs to the extent of 75 pmole binding per nmole pentachlorophenol. This is almost enough to account for the amount of label bound to protein observed in hexachlorobenzene incubations. This indicates that less than 10% of the covalent binding occurs during conversion of hexachlorobenzene to pentachlorophenol, and the remainder is produced during conversion of pentachlorophenol. The major product of musomal oxidation of pentachlorophenol is tetrachlorohydroquinone, which is in redox-equilibrium with the corresponding semiquinone and quinone (chloranil). The covalent binding is inhibited by addition of ascorbic acid or glutathione to the hexachlorobenze incubations. Ascorbic acid decreases the covalent binding with a simultaneous increase in formation of tetrachlorohydroquinone, probably due to a shift in the redox-equilibrium to the reduced side. Glutathione does not act as a reducing agent, since the inhibition of covalent binding is not accompanied by an increase in tetrachlorohydroquinone formation. Instead, glutathione reacts with chloranil, producing at least three stable products, probably in a Michael-type reaction. These results strongly indicate the involvement of chloranil or the semiquinone radical in the covalent binding during musomal hexachlorobenzene metabolism.

Characteristics of the active oxygen in covalent binding of the pesticide methoxychlor to hepatic microsomal proteins

This study examined the characteristics of the active oxygen species involved in generation of the reactive intermediate of methoxychlor which covalently binds to liver microsomal proteins. The possibility that the active oxygen participating in the above reaction is the Superoxide anion (O − 2) or a species generated from O − 2 was examined with the help of Superoxide dismutase (SOD) and with an SOD-mimetic agent, CuDIPS [Cu 2+(3,5-diisopropylsalicyclic acid)2]. It was observed that, whereas CuDIPS inhibited covalent binding of methoxychlor metabolite(s), SOD did not. However, ZnDIPS [Zn 2+(3,5-diisopropylsalicyclic acid);], which exhibits no SOD-mimetic activity, did not inhibit covalent binding. Furthermore, both CuDIPS and ZnDIPS had little or no effect on the formation of demethylated (polar) metabolites of methoxychlor, demonstrating that the inhibition of covalent binding by CuDIPS was not merely due to a general inhibition of the hepatic monooxygenase system. These findings suggested that O 2 − was involved in covalent binding, but was not accessible to SOD. Additional support for O 2 − involvement stems from the observation that a-tocopheryl acid succinate markedly inhibited covalent binding of methoxychlor. The possibility that hydrogen peroxide (H 2O 2) was involved in covalent binding of methoxychlor appears unlikely. Catalase had no effect on covalent binding when NADPH was the cofactor, and the use of H 2O 2 in place of NADPH did not yield covalent binding. Certain scavengers of hydroxyl radical (ethanol, t-butanol and benzoate) inhibited, and other known scavengers (DMSO and mannitol) did not inhibit, covalent binding. EDTA stimulated binding, desferal (desferrioxamine) exhibited no effect on binding, and diethylenetriaminepentaacetic acid (DETAPAC) inhibited binding. A possible explanation for this observation is that the Fe 2− needed for generation of OH is much more easily obtained from Fe 3+-EDTA than from Fe 3+-desferal, which resists reduction. The inhibitory effect by DETAPAC may be due to chelation of another metal which is needed for the reaction. Lastly, certain scavengers of singlet oxygen inhibited covalent binding with little effect on the formation of polar metabolites of methoxychlor. In conclusion, these studies support the involvement of OH and singlet oxygen, possibly derived from O − 2, in the formation of the reactive methoxychlor intermediate. However, the less likely possibility that these compounds, used to probe for active oxygen, merely inhibit the activity of a cytochrome P-450 which specifically catalyzes the formation of the reactive methoxychlor intermediate but not of the demethylation of methoxychlor cannot be ruled out.

Metabolism of 1-naphthol by tyrosinase

1-Naphthol was metabolized by the polyphenol oxidase, tyrosinase, primarily to 1,2-naphthoquinone and to small amounts of 1,4-naphthoquinone as well as to covalently bound products. The inhibition of covalent binding by ethylenediamine, which reacts specifically with 1,2-naphthoquinone but not 1,4-naphthoquinone, suggested that most of the covalent binding was due to 1,2-naphthoquinone or a metabolite of similar structure. The activation by tyrosinase of 1-naphthol to covalently bound products suggested that it may alter the reaction kinetics of the enzyme. This was investigated by studying the effects of 1-naphthol on the tyrosinase-catalysed oxidation of 4-hydroxy anisole. Preincubation of tyrosinase with 1-naphthol increased the lag period of the oxidation of 4-hydroxyanisole, which may be due to a decrease in the amount of active enzyme, as well as to a reaction of 1-naphthol with 3,4-anisylquinone, an oxidation product of 4-hydroxyanisole. The metabolic activation of 1-naphthol by tyrosinase to covalently bound species suggests that 1-naphthol or a structurally related derivative may be of potential therapeutic application in the treatment of cells high in tyrosinase activity, such as certain melanomas.