ABSTRACT
In this study, the new stable sulfur-containing compounds onionins A2 (1) and A3 (2) were isolated from the acetone extracts of the bulbs of Allium cepa L. and identified as the stereoisomers of onionin A1 discovered in our previous study. Their chemical structures, 3,4-dimethyl-5-(1E-propenyl)-tetrahydrothiophene-2-sulfenic acid-S-oxides, were characterized using various spectroscopic techniques. In addition, 1 and 2 together with onionin A1 were successfully isolated from the leaves of the Welsh onion, Allium fistulosum L. The onion-extracted fractions showed good potential to inhibit the polarization of M2 activated macrophages, indicating their possible ability to inhibit tumor cell proliferation.
Subject(s)
Immunologic Factors/chemistry , Macrophages/drug effects , Onions/chemistry , Sulfenic Acids/chemistry , Thiophenes/chemistry , Antigens, CD/analysis , Antigens, CD/immunology , Antigens, Differentiation, Myelomonocytic/analysis , Antigens, Differentiation, Myelomonocytic/immunology , Antineoplastic Agents, Phytogenic/chemistry , Antineoplastic Agents, Phytogenic/isolation & purification , Antineoplastic Agents, Phytogenic/pharmacology , Cells, Cultured , Humans , Immunologic Factors/isolation & purification , Immunologic Factors/pharmacology , Macrophages/immunology , Plant Roots/chemistry , Receptors, Cell Surface/analysis , Receptors, Cell Surface/immunology , Sulfenic Acids/isolation & purification , Sulfenic Acids/pharmacology , Thiophenes/isolation & purification , Thiophenes/pharmacologyABSTRACT
Facile, two-step synthesis and kinetic characterization of new chemical probes for selective labeling of sulfenic acid (-SOH) in proteins are presented. The synthesis route relies on the simple and highly efficient Michael addition of thiol containing tags or linkers to 4-cyclopentene-1,3-dione, the unsaturated derivative of 1,3-cyclopentanedione.
Subject(s)
Cyclopentanes/chemical synthesis , Proteins/chemistry , Sulfenic Acids/chemistry , Animals , Cyclopentanes/chemistry , Mice , NIH 3T3 Cells , Proteins/isolation & purification , Staining and Labeling/methods , Sulfenic Acids/isolation & purification , Sulfhydryl Compounds/chemical synthesis , Sulfhydryl Compounds/chemistryABSTRACT
Human serum albumin (HSA) is the most abundant protein in the intravascular compartment. It possesses a single thiol, Cys34, which constitutes ~80 percent of the total thiols in plasma. This thiol is able to scavenge plasma oxidants. A central intermediate in this potential antioxidant activity of human serum albumin is sulfenic acid (HSA-SOH). Work from our laboratories has demonstrated the formation of a relatively stable sulfenic acid in albumin through complementary spectrophotometric and mass spectrometric approaches. Recently, we have been able to obtain quantitative data that allowed us to measure the rate constants of sulfenic acid reactions with molecules of analytical and biological interest. Kinetic considerations led us to conclude that the most likely fate for sulfenic acid formed in the plasma environment is the reaction with low molecular weight thiols to form mixed disulfides, a reversible modification that is actually observed in ~25 percent of circulating albumin. Another possible fate for sulfenic acid is further oxidation to sulfinic and sulfonic acids. These irreversible modifications are also detected in the circulation. Oxidized forms of albumin are increased in different pathophysiological conditions and sulfenic acid lies in a mechanistic junction, relating oxidizing species to final thiol oxidation products.
Subject(s)
Humans , Serum Albumin/chemistry , Serum Albumin/metabolism , Sulfenic Acids/metabolism , Sulfhydryl Compounds/metabolism , Oxidation-Reduction , Protein Conformation , Sulfenic Acids/isolation & purificationABSTRACT
Human serum albumin (HSA) is the most abundant protein in the intravascular compartment. It possesses a single thiol, Cys34, which constitutes ~80% of the total thiols in plasma. This thiol is able to scavenge plasma oxidants. A central intermediate in this potential antioxidant activity of human serum albumin is sulfenic acid (HSA-SOH). Work from our laboratories has demonstrated the formation of a relatively stable sulfenic acid in albumin through complementary spectrophotometric and mass spectrometric approaches. Recently, we have been able to obtain quantitative data that allowed us to measure the rate constants of sulfenic acid reactions with molecules of analytical and biological interest. Kinetic considerations led us to conclude that the most likely fate for sulfenic acid formed in the plasma environment is the reaction with low molecular weight thiols to form mixed disulfides, a reversible modification that is actually observed in ~25% of circulating albumin. Another possible fate for sulfenic acid is further oxidation to sulfinic and sulfonic acids. These irreversible modifications are also detected in the circulation. Oxidized forms of albumin are increased in different pathophysiological conditions and sulfenic acid lies in a mechanistic junction, relating oxidizing species to final thiol oxidation products.
Subject(s)
Serum Albumin/chemistry , Serum Albumin/metabolism , Sulfenic Acids/metabolism , Sulfhydryl Compounds/metabolism , Humans , Oxidation-Reduction , Protein Conformation , Sulfenic Acids/isolation & purificationABSTRACT
Sulfenic acid (RSOH) is a central intermediate in both the reversible and irreversible redox modulation by reactive species of an increasing number of proteins involved in signal transduction and enzymatic pathways. In this paper we focus on human serum albumin (HSA), the most abundant plasma protein, proposed to serve antioxidant functions in the vascular compartment. Sulfenic acid in HSA has been previously detected using different methods after oxidation of its single free thiol Cys34 through one- or two-electron mechanisms. Since recent evidence suggests that sulfenic acid in HSA is stabilized within the protein environment, this derivative represents an appropriate model to examine protein sulfenic acid biochemistry, structure and reactivity. Sulfenic acid in HSA could be involved in mixed disufide formation, supporting a role of HSA-Cys34 as an important redox regulator in extracellular compartments.
Subject(s)
Free Radicals/metabolism , Serum Albumin/chemistry , Serum Albumin/metabolism , Sulfenic Acids/metabolism , Humans , Models, Molecular , Oxidants , Oxidation-Reduction , Protein Conformation , Sulfenic Acids/isolation & purificationABSTRACT
The trapping of a sulfenic acid within the fully active C165S mutant of the AhpC peroxidase protein from Salmonella typhimurium was investigated. The electrophilic reagent employed in these studies, 7-chloro-4-nitrobenz-2-oxa-1,3-diazole (NBD-Cl), has previously been used to modify thiol, amino, and tyrosine hydroxyl groups in proteins; at neutral pH only cysteinyl residues of AhpC proteins are modified. The peroxide-oxidized C165S mutant of AhpC incubated with NBD-Cl gave a product with an absorbance maximum at 347 nm, whereas the thiol-NBD conjugate formed from the reduced protein absorbed maximally at 420 nm. Electrospray ionization mass spectrometry of the modified proteins allowed identification of the species absorbing at 347 nm as a Cys-S(O)-NBD derivative containing one additional oxygen relative to the Cys-S-NBD product. The C165S conjugates with Cys-S(O)-NBD and Cys-S-NBD had no peroxidase activity when compared to unreacted C165S and wild-type AhpC, but were both reactivated through removal of NBD by DTT. Oxidized C165S was also modified by dimedone, a common sulfenic acid reagent, to give the expected inactivated conjugate of higher mass. This reagent was not removed by DTT and blocked any further reaction of the protein with NBD-Cl. NBD modification of Enterococcus faecalis NADH peroxidase, a well-characterized flavoprotein with an active-site sulfenic acid (Cys-SOH), also yielded the spectrally-distinguishable NBD conjugates following incubation of NBD-Cl with oxidized and reduced forms of the denatured peroxidase, indicating a general utility for this reagent with other sulfenic acid-containing proteins. A significant advantage of NBD-Cl over previously-used sulfenic acid reagents such as dimedone is in the retention of the sulfenic acid oxygen in the modified product; differentiation between protein-associated thiols and sulfenic acids is therefore now possible by means of both visible absorbance properties and mass analyses of the NBD-modified proteins.
