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Aldose reductase (ALR2) is susceptible to oxidative inactivation by copper ion. The mechanism underlying the reversible modification of ALR2 was studied by mass spectrometry, circular dichroism, and molecular modeling approaches on the enzyme purified from bovine lens and on wild type and mutant recombinant forms of the human placental and rat lens ALR2. Two equivalents of copper ion were required to inactivate ALR2: one remained weakly bound to the oxidized protein whereas the other was strongly retained by the inactive enzyme. Cys303 appeared to be the essential residue for enzyme inactivation, because the human C303S mutant was the only enzyme form tested that was not inactivated by copper treatment. The final products of human and bovine ALR2 oxidation contained the intramolecular disulfide bond Cys298-Cys303. However, a Cys80-Cys303 disulfide could also be formed. Evidence for an intramolecular rearrangement of the Cys80-Cys303 disulfide to the more stable product Cys298-Cys303 is provided. Molecular modeling of the holoenzyme supports the observed copper sequestration as well as the generation of the Cys80-Cys303disulfide. However, no evidence of conditions favoring the formation of the Cys298-Cys303 disulfide was observed. Our proposal is that the generation of the Cys298-Cys303 disulfide, either directly or by rearrangement of the Cys80-Cys303 disulfide, may be induced by the release of the cofactor from ALR2 undergoing oxidation. The occurrence of a less interactive site for the cofactor would also provide the rationale for the lack of activity of the disulfide enzyme forms.

Glutathione is one of the most relevant antioxidants present in cells. It exerts its scavenging action through the involvement of efficient and ubiquitous enzymes. GSH on the other hand, because of its chemical features, can scavenge reactive oxygen species without the involvement of enzymatic systems. The study deals with the mobilization of GSH pool in a nonenzymatic antioxidant system by other physiological thiols (i.e., cysteine and cysteinyl-glycine), which are far more sensitive than GSH to oxidative conditions. These thiol compounds, in the presence of iron/EDTA, can promote oxygen activation through their oxidation to disulfides. GSH, through trans-thiolation reactions, can regenerate Cys and CysGly, which can then recycle, thus inducing a massive GSH oxidation. In these conditions, making use of bovine lens aldose reductase as a protein model, evidence is given that Cys and CysGly promote specific protein S-thiolation reactions. The possibility that GSH may be recruited in controlling cellular oxygen tension is considered.

The glutathionyl-modified aldose reductase (GS-ALR2) is unique, among different S-thiolated enzyme forms, in that it displays a lower specific activity than the native enzyme (ALR2). Specific interactions of the bound glutathionyl moiety (GS) with the ALR2 active site, were predicted by a low perturbative molecular modelling approach. The outcoming GS allocation, involving interactions with residues relevant for catalysis and substrate allocation, explains the rationale behind the observed differences in the activity between GS-ALR2 and other thiol-modified enzyme forms. The reversible S-glutathionylation of ALR2 observed in cultured intact bovine lens undergoing an oxidative/non oxidative treatment cycle is discussed in terms of the potential of ALR2/GS-ALR2 inter-conversion as a response to oxidative stress conditions.

The reversibility of S-thiolation of aldose reductase was shown in intact bovine lens subjected to oxidative stress. The glutathione modified aldose reductase generated in the lens as a consequence of hyperbaric oxygen treatment was recovered in its reduced form following culturing in normobaric air conditions. Nucleus and cortex were differently affected by both oxidative treatment and normobaric air recovery. The extent of S-thiolation of aldose reductase appeared to be higher in the nucleus than in the cortex. Moreover, the nucleus, but not the cortex, was unable to completely recover from the protein S-thiolation process. The ratios of GSH/GSSG and NADPH/NADP+as well as the Energy Charge values were determined in the cortex and nucleus both after oxidative stress and recovery. The results are consistent with the existence of a quite well-defined boundary between the two lens regions. Moreover, they are supportive of the hypothesis that thiol/disulfide exchange has the potential to be a regulatory mechanism for certain enzymes which can modulate the flux of NADPH inside the cell.

The occurrence of protein S-thiolation as a consequence of oxidative stress is widely recognized (Thomas et al., 1990; Lou et at, 1990; Schuppe et al., 1992; Giblin, et al., 1995). However, the role and the relevance of this process are still a matter of debate.

A study on the kinetics of human thrombin inhibition by two novel synthetic peptides (Hirunorm IV and Hirunorm V) and a comparison with recombinant hirudin and a commonly used thrombin inhibitor, Hirulog-1, are reported. The dissociation constants for Hirunorm IV and Hirunorm V were determined by varying the concentration of inhibitors at fixed concentrations of the chromogenic substrate Chromozym-TH (N-tosylglycyl-L-prolyl-L-arginine 4-nitroanilide acetate). Both inhibitors behaved as reversible tight-binding inhibitors of amidolytic thrombin activity. The apparent dissociation constants determined showed a linear dependence on the concentration of substrate; this finding, which indicates that the inhibition was competitive, made possible the estimation of the dissociation constants ( K I ) for Hirunorm IV and Hirunorm V, which were 0.134 ± 0.014 nM and 0.245 ± 0.016 nM, respectively. Similar dissociation constants were also obtained for the two inhibitors when thrombin activity was measured with fibrinogen in the clotting assay. When tested for resistance to thrombin proteolytic activity, both inhibitors were inviolate to cleavage by thrombin. The data obtained demonstrate that both Hirunorm IV and Hirunorm V are potent and stable inhibitors of human thrombin activity.

Oxidative stress is one of the most deleterious conditions for cell systems. A number of oxidative modification processes involving highly relevant biological molecules can be seen as primary events in the development of pathological states which may lead, depending on the severity of the oxidative insult, to cell death [1–5]. Among others, thiols represent an important class of molecules which are easily involved in redox processes. This because of their generally high susceptibility to oxidation which may occur in biological systems at relatively high rate without the assistance of enzymatic systems. Moreover, the potential reversibility of at least the initial steps of thiol oxidation (i.e. thiol to disulfide) and the presence of thiols on proteins give to these functional groups the feature to directly connect oxidative phenomena with the molecular machinery responsible for the metabolic control. Another emerging aspect of thiol/disulfide interconversion is the paradoxical prooxidant ability of thiol molecules [6–11] whose oxidation, linked to O2/H2O2 interconversion, may be associated to cell signalling phenomena [12–16]. Indeed, the scavenging action exerted by low molecular weight thiols in counteracting oxidative stress conditions can be amplified via the S-thiolation of enzymes [17–19]. However, whether S-thiolation is a way to control enzyme activity or is the unavoidable consequence of the reactivity of thiols is still a matter of debate, essentially because of the lack of specialised enzymes able to catalyze thiolation/de-thiolation reactions. In addition, even though the reaction rates of non-enzymatic S-thiolation processes are sufficiently high to be compatible with cellular functions, the problem linked to the required specificity of the modification stands. In this regard, however, it should be considered that the target molecules are highly structurally organized. Thus the restraint in the accessibility of specific thiolating agents into the thiolation site, combined with their peculiar effects on the target protein, may impose sufficient restrictions to confer to the process features of specificity. However, it is also true that the body of data on enzymes able to intervene into the protein thiol/disulfide interconversion is growing [20–23], so that the lack of enzymatic driven S-thiolation processes may only be a temporary lack of knowledge.