Thiols (RSH) are potent nucleophilic agents, the rates of which depend on the pKa of the sulfhydryl. Unlike compounds having other nucleophile moieties (-OH or -NH(2)), RSH are involved in reactions, such as conjugations, redox and exchange reactions. Although protein SH groups (PSH) react like non-protein thiols (NPSH), the biochemistry of proteins is much more complex for reasons such as steric hindrance, charge distribution and accessibility of PSH to the solvent (protein conformation). The reaction rates and types of end-products of PSH vary a lot from protein to protein. The biological problem is even more complex because in all compartments and tissues, there may be specific competition between thiols (namely between GSH and PSH), regulated by the properties of antioxidant enzymes. Moreover, PSH are divided biologically into essential and non-essential and their respective influence in the various biological systems is unknown. It follows that during phenomena eliciting a prompt thiol response (oxidative stress), the antioxidant PSH response and reaction mechanisms vary considerably from case to case. For example, in spite of a relatively low pKa that should guarantee good antioxidant capacity, PSH of albumin has much less propensity to form adducts with conjugating agents than NPSH; moreover, the structural characteristics of the protein prevent albumin from forming protein disulfides when exposed to oxidants (whereas protein-thiol mixed disulfides are formed in relative abundance). On the other hand, proteins with a relatively high reactivity, such rat hemoglobin, have much greater antioxidant capacity than GSH, but although human hemoglobin has a pKa similar to GSH, for structural reasons it has less antioxidant capacity than GSH. When essential PSH are involved in S-thiolation and S-nitrosation reactions, a similar change in biological activity is observed. S-thiolated proteins are a recurrent phenomenon in oxidative stress elicited by reactive oxygen species (ROS). This event may be mediated by disulfides, that exchange with PSH, or by the protein intermediate sulfenic acid that reacts with thiols to form protein-mixed disulfides. During nitrosative stress elicited by reactive nitrogen species (RNS), depending on the oxygen concentration of the system, nitrosation reactions of thiols may also be accompanied by protein S-thiolation. In this review we discuss a number of cell processes and biochemical modifications of enzymes that indicate that S-thiolation and S-nitrosation may occur simultaneously in the same protein in the presence of appropriate interactions between ROS and RNS.
We previously demonstrated a high susceptibility of neonatal red blood cells (RBC) to oxidative stress at birth. The aim of this study was to compare the RBC antioxidant capacity and redox cycle enzyme activities as well as glutathione (GSH) recycling in full-term and preterm infants at birth and in normal adults. GSH and GSH disulfide (GSSG) concentrations, GSH/GSSG ratio, and the activities of glucose-6-phosphate dehydrogenase (G-6-PDH), GSH peroxidase, GSH reductase (GR), catalase (CAT), superoxide dismutase (SOD), and hexokinase (HK) were measured in RBC of 25 healthy adults and 56 newborns (23 term, 33 preterm) at birth. The GSH recycling was measured in adult and newborn RBC exposed to oxidative stress (1 mM tert-butylhydroperoxide). The RBC of term and preterm babies showed higher GSH, GSSG, G-6-PDH, GR, and HK levels/activities and lower GSH/GSSG ratios and higher GSH-recycling rates than those of adults. In preterm babies significant correlations were found between G-6-PDH and CAT, GSH, GSH/GSSG ratio, and GSSG (r = –0.67, r = 0.71, r = –0.66, p < 0.01; r = 0.71, p < 0.05, respectively). In term newborns, statistically significant correlations were observed between G-6-PDH and CAT, SOD, and GSH (r = –0.65, r = –0.65, r = –0.69, p < 0.01, respectively). The results indicate the central role of the G-6-PDH activity in antioxidant defenses. We speculate that preterm babies have prompter involvement of antioxidant defenses than term babies.
Dethiolation experiments of thiolated albumin with thionitrobenzoic acid and thiols (glutathione, cysteine, homocysteine) were carried out to understand the role of albumin in plasma distribution of thiols and disulfide species by thiol/disulfide (SH/SS) exchange reactions. During these experiments we observed that thiolated albumin underwent thiol substitution (Alb-SS-X+RSH<-->Alb-SS-R+XSH) or dethiolation (Alb-SS-X+XSH<-->Alb-SH+XSSX), depending on the different pK(a) values of thiols involved in protein-thiol mixed disulfides (Alb-SS-X). It appeared in these reactions that the compound with lower pK(a) in mixed disulfide was a good leaving group and that the pK(a) differences dictated the kind of reaction (substitution or dethiolation). Thionitrobenzoic acid, bound to albumin by mixed disulfide (Alb-TNB), underwent rapid substitution after thiol addition, forming the corresponding Alb-SS-X (peaks at 0.25-1 min). In turn, Alb-SS-X were dethiolated by the excess nonprotein SH groups because of the lower pK(a) value in mixed disulfide with respect to that of other thiols. Dethiolation of Alb-SS-X was accompanied by formation of XSSX and Alb-SH up to equilibrium levels at 35 min, which were different for each thiol. Structures by molecular simulation of thiolated albumin, carried out for understanding the role of sulfur exposure in mixed disulfides in dethiolation process, evidenced that the sulfur exposure is important for the rate but not for determining the kind of reaction (substitution or dethiolation). Our data underline the contribution of SH/SS exchanges to determine levels of various thiols as reduced and oxidized species in human plasma.
Protein thiolation is elicited by oxidation by different mechanisms and is involved in a variety of biological processes. Thiols, protein SH (PSH) and non-protein SH groups (NPSH, namely GSH), are in competition in all biological environments in the regulation of oxidant homeostasis because oxidants thiolate proteins, whereas GSH dethiolates them (e.g., GSSG + PSH --> GSSP + GSH). Although poorly investigated, the elimination of disulfides from thiolated proteins to regenerate critical PSH is important. These aspects are poorly known in cells, where glutaredoxin and peroxiredoxin operate as enzymes or potential chaperones to accelerate dethiolation. On the contrary, studies with plasma or albumin have highlighted the importance of protein conformation in dethiolation processes and have clarified the reason why homocysteine (thiol with potential toxicity) is preferentially bound to albumin as protein-thiol mixed disulfide with respect to other NPSH. Here we provide an overview of protein thiolation/dethiolation processes, with an emphasis on recent developments and future perspectives in this field.
We investigated the effect of extra virgin olive oil (EVOO) on platelet aggregation and plasma concentrations of homocysteine (Hcy) redox forms in rats in relation to the minor polar compound (MPC) concentration of EVOO. We used 3 olive oil samples with similar fatty acid but different MPC concentrations: refined olive oil (RF) with traces of MPC (control oil), native EVOO with low MPC concentration (LC), and EVOO with high MPC concentration (HC) enriching LC with its own MPC. Oil samples were administered to rats by gavage (1.25 mL/kg body weight) using 2 experimental designs: acute (24-h food deprivation and killed 1 h after EVOO administration) and subacute (12-d treatment, a daily dose of oil for 12 d, and killed after 24 h of food deprivation). Platelet aggregation was induced by ADP (ex vivo tests) and a reduction in platelet reactivity occurred in cells from rats given LC in the subacute study and in cells from rats administered HC in both studies as indicated by an increase in the agonist half maximal effective concentration. HC inhibited platelet aggregation induced by low ADP doses (reversible aggregation) in cells of rats in both the acute and subacute studies, whereas LC had this effect only in the subacute experiment. Moreover, in rats administered HC in both experiments, the plasma concentration of free reduced Hcy (rHcy) was lower and Hcy bound to protein by disulfide bonds (bHcy) was greater than in RF-treated rats. bHcy was also greater in rats given LC than in RF-treated rats in the subacute experiment. Plasma free-oxidized Hcy was greater in rats given LC and HC than in those administered RF only in the subacute experiment. In conclusion, these results show that MPC in EVOO inhibit platelet aggregation and reduce the plasma rHcy concentration, effects that may be associated with cardiovascular protection.
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