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• W2016553301 abstract "The S-conjugation rates of the free-reacting thiols present on each component of rat hemoglobin with 5,5-dithio-bis(2,2-nitrobenzoic acid) (DTNB) have been studied under a variety of conditions. On the basis of their reactivity with DTNB (0.5 mm), three classes of thiols have been defined as follows: fast reacting (fHbSH), with t½ <100 ms; slow reacting (sHbSH), with t½ 30–50 s; and very slow reacting (vsHbSH), with t½ 180–270 s. Under paraphysiological conditions, fHbSH (identified with Cys-125β(H3)) conjugates with DTNB 100 times faster than glutathione and ∼4000 times more rapidly than (v)sHbSH (Cys-13α(A11) and Cys-93β(F9)). Such characteristics of fHbSH reactivity that are independent of the quaternary state of hemoglobin are mainly due to the following: (i) its low pK (∼6.9, the cysteinyl anion being stabilized by a hydrogen bond with Ser-123β(H1)) and (ii) the large exposure to the solvent (as measured by analysis of a model of the molecular surface) and make these thiols the kinetically preferred groups in rat erythrocytes for S-conjugation. In addition, because of the high cellular concentration (8 mm, i.e. four times that of glutathione), fHbSHs are expected to intercept damaging species in erythrocytes more efficiently than glutathione, thus adding a new physiopathological role (direct involvement in cellular strategies of antioxidant defense) to cysteinyl residues in proteins. The S-conjugation rates of the free-reacting thiols present on each component of rat hemoglobin with 5,5-dithio-bis(2,2-nitrobenzoic acid) (DTNB) have been studied under a variety of conditions. On the basis of their reactivity with DTNB (0.5 mm), three classes of thiols have been defined as follows: fast reacting (fHbSH), with t½ <100 ms; slow reacting (sHbSH), with t½ 30–50 s; and very slow reacting (vsHbSH), with t½ 180–270 s. Under paraphysiological conditions, fHbSH (identified with Cys-125β(H3)) conjugates with DTNB 100 times faster than glutathione and ∼4000 times more rapidly than (v)sHbSH (Cys-13α(A11) and Cys-93β(F9)). Such characteristics of fHbSH reactivity that are independent of the quaternary state of hemoglobin are mainly due to the following: (i) its low pK (∼6.9, the cysteinyl anion being stabilized by a hydrogen bond with Ser-123β(H1)) and (ii) the large exposure to the solvent (as measured by analysis of a model of the molecular surface) and make these thiols the kinetically preferred groups in rat erythrocytes for S-conjugation. In addition, because of the high cellular concentration (8 mm, i.e. four times that of glutathione), fHbSHs are expected to intercept damaging species in erythrocytes more efficiently than glutathione, thus adding a new physiopathological role (direct involvement in cellular strategies of antioxidant defense) to cysteinyl residues in proteins. Human but not rat erythrocytes are reported (1Kosower N.S. Kosower E.M. Dolphin D. Poulson R. Avramovic O. Coenzymes and Cofactors. Vol. III. John Wiley & Sons, Inc., New York1989: 319-356Google Scholar) to be able to restore the cellular pool of GSH, which is strongly decreased after treatment with diazenedicarboxylic acid bis(N,N-dimethylamide), a thiol-oxidizing agent known by the trivial name diamide (2Kosower N.S. Kosower E.M. Methods Enzymol. 1995; 251: 123-133Crossref PubMed Scopus (284) Google Scholar). Such a difference in redox behavior was attributed to a lower enzymatic capacity in reducing disulfides of rat red cells relative to the human ones (3Kosower N.S. Kosower E.M. Koppel R.L. Eur. J. Biochem. 1977; 77: 529-534Crossref PubMed Scopus (42) Google Scholar). However, recent work in this laboratory demonstrated that the reversibility of such process can be observed also in rat erythrocytes, depending on diamide dose (4Di Simplicio P. Lupis E. Rossi R. Biochim. Biophys. Acta. 1996; 1289: 257-275Google Scholar). Additional evidence (3Kosower N.S. Kosower E.M. Koppel R.L. Eur. J. Biochem. 1977; 77: 529-534Crossref PubMed Scopus (42) Google Scholar) may suggest that these differences in behavior between rat and human erythrocytes could be related to diversities in reactivity of sulfhydryl groups of the corresponding hemoglobins.Studies of the reactivities of the sulfhydryl groups of oxygen carriers (5Garel M.C. Beuzard Y. Thillet J. Domenget C. Martin J. Galacteros F. Rosa J. Eur. J. Biochem. 1982; 123: 513-519Crossref PubMed Scopus (47) Google Scholar, 6Taylor J.F. Antonini E. Brunori M. Wyman J. J. Biol. Chem. 1966; 241: 241-248Abstract Full Text PDF PubMed Google Scholar, 7Birchmeier W. Tuchschmid P.E. Winterhalter K. Biochemistry. 1973; 12: 3667-3672Crossref PubMed Scopus (22) Google Scholar, 8Garel M.C. Domenget C. Caburi-Martin J. Prehu C. Galacteros F. Beuzard Y. J. Biol. Chem. 1986; 261: 14704-14709Abstract Full Text PDF PubMed Google Scholar) are mainly restricted to hemoglobins containing only two accessible thiols per tetrameric molecule (such as human hemoglobin), located at the (F9)93 position of each β subunit. Many sulfhydryl reagents can be bound to Cys-93β(F9), even in intact red blood cells (8Garel M.C. Domenget C. Caburi-Martin J. Prehu C. Galacteros F. Beuzard Y. J. Biol. Chem. 1986; 261: 14704-14709Abstract Full Text PDF PubMed Google Scholar). Nevertheless, despite its high intracellular level (about half the concentration of the hemoglobin tetramer (9Beutler E. Red Cell Metabolism: A Manual of Biochemical Methods. 2nd Ed. Grune and Stratton, Inc., Orlando, FL1975: 105-107Google Scholar)), glutathione is not significantly combined with this protein-bound thiol under normal conditions. GSSG in fact is reported (5Garel M.C. Beuzard Y. Thillet J. Domenget C. Martin J. Galacteros F. Rosa J. Eur. J. Biochem. 1982; 123: 513-519Crossref PubMed Scopus (47) Google Scholar, 6Taylor J.F. Antonini E. Brunori M. Wyman J. J. Biol. Chem. 1966; 241: 241-248Abstract Full Text PDF PubMed Google Scholar, 7Birchmeier W. Tuchschmid P.E. Winterhalter K. Biochemistry. 1973; 12: 3667-3672Crossref PubMed Scopus (22) Google Scholar, 8Garel M.C. Domenget C. Caburi-Martin J. Prehu C. Galacteros F. Beuzard Y. J. Biol. Chem. 1986; 261: 14704-14709Abstract Full Text PDF PubMed Google Scholar) (i) to react very slowly with human hemoglobin in vitro and (ii) to spontaneously form with this protein adduct in vivo only under very special conditions and in small amounts (2–7% of the total hemoglobin), having been observed to date solely in hemolysates of patients on long term anti-epileptic therapy (10Niketic V. Beslo D. Raicevic S. Sredic S. Tojkovic M. Int. J. Biochem. 1992; 24: 503-507Crossref PubMed Scopus (10) Google Scholar). On the contrary, evidence exists that rat erythrocytes produce large quantities of glutathione-protein mixed disulfides under any oxidative stress (4Di Simplicio P. Lupis E. Rossi R. Biochim. Biophys. Acta. 1996; 1289: 257-275Google Scholar,11Di Simplicio P. Rossi R. Biochim. Biophys. Acta. 1994; 1199: 245-252Crossref PubMed Scopus (29) Google Scholar, 12Terada T. Nishimura M. Oshida H. Oshida T. Mizoguchi T. Biochem. Mol. Biol. Int. 1993; 29: 1009-1014PubMed Google Scholar, 13Grossman S.J. Jollow D.J. J. Pharmacol. Exp. Ther. 1987; 244: 118-125Google Scholar), most of them being adducts with hemoglobin thiols (HbSSG). 1The abbreviations used are: HbSSG, glutathione-hemoglobin mixed disulfide; t-BOOH,tert-butylhydroperoxide; DTNB, 5,5-dithio-bis(2-nitrobenzoic acid); HbSSHb, hemoglobin-hemoglobin disulfides; HbSH, reactive sulfhydryl groups on hemoglobin; fHbSH, fast reacting sulfhydryl groups on rat hemoglobin components; vsHbSH, slow reacting sulfhydryl groups on rat hemoglobin components; vsHbSH, very slow reacting sulfhydryl groups on rat hemoglobin components; (v)sHbSH, very slow reacting and/or slow reacting sulfhydryl groups on rat hemoglobin components; HPLC, high pressure liquid chromatography. 1The abbreviations used are: HbSSG, glutathione-hemoglobin mixed disulfide; t-BOOH,tert-butylhydroperoxide; DTNB, 5,5-dithio-bis(2-nitrobenzoic acid); HbSSHb, hemoglobin-hemoglobin disulfides; HbSH, reactive sulfhydryl groups on hemoglobin; fHbSH, fast reacting sulfhydryl groups on rat hemoglobin components; vsHbSH, slow reacting sulfhydryl groups on rat hemoglobin components; vsHbSH, very slow reacting sulfhydryl groups on rat hemoglobin components; (v)sHbSH, very slow reacting and/or slow reacting sulfhydryl groups on rat hemoglobin components; HPLC, high pressure liquid chromatography. In particular, treatment of rat red cells with t-BOOH leads to GSH oxidation with formation of both GSSG and HbSSG, followed by a recovery of the initial values within 60 min; on the other hand, addition of diamide to the same system only causes an increase in HbSSG with no formation of GSSG (4Di Simplicio P. Lupis E. Rossi R. Biochim. Biophys. Acta. 1996; 1289: 257-275Google Scholar).All these facts, as a whole, prompted us to investigate further some structural and functional properties of rat hemoglobin thiols. Many hemoglobin components (6Taylor J.F. Antonini E. Brunori M. Wyman J. J. Biol. Chem. 1966; 241: 241-248Abstract Full Text PDF PubMed Google Scholar, 7Birchmeier W. Tuchschmid P.E. Winterhalter K. Biochemistry. 1973; 12: 3667-3672Crossref PubMed Scopus (22) Google Scholar, 8Garel M.C. Domenget C. Caburi-Martin J. Prehu C. Galacteros F. Beuzard Y. J. Biol. Chem. 1986; 261: 14704-14709Abstract Full Text PDF PubMed Google Scholar, 9Beutler E. Red Cell Metabolism: A Manual of Biochemical Methods. 2nd Ed. Grune and Stratton, Inc., Orlando, FL1975: 105-107Google Scholar, 10Niketic V. Beslo D. Raicevic S. Sredic S. Tojkovic M. Int. J. Biochem. 1992; 24: 503-507Crossref PubMed Scopus (10) Google Scholar) have been separated from hemolysates of adult rats (14Stein S. Cherian J. Mazur A. J. Biol. Chem. 1971; 246: 5287-5293Abstract Full Text PDF PubMed Google Scholar, 15Garrik L.M. Sharma S.V. McDonald M.J. Ranney H.M. Biochem. J. 1975; 149: 245-258Crossref PubMed Scopus (65) Google Scholar, 16Datta M.C. Gilman J.G. Hemoglobin. 1981; 5: 701-714Crossref PubMed Scopus (22) Google Scholar), and two types of α subunits and four types of β subunits have been identified (17Ferranti P. Carbone V. Sannolo N. Fiume I. Malorni A. Int. J. Biochem. 1993; 25: 1943-1950Crossref PubMed Scopus (20) Google Scholar). The primary structures reveal the presence of 3 cysteinyl residues on the major α chain (All(13), G11(104), and G18(111)) and up to 2 on the β chain (F9(93) common to all 4 β subunits, and H3(125) in 3 out of 4 β subunits) (15Garrik L.M. Sharma S.V. McDonald M.J. Ranney H.M. Biochem. J. 1975; 149: 245-258Crossref PubMed Scopus (65) Google Scholar, 16Datta M.C. Gilman J.G. Hemoglobin. 1981; 5: 701-714Crossref PubMed Scopus (22) Google Scholar, 17Ferranti P. Carbone V. Sannolo N. Fiume I. Malorni A. Int. J. Biochem. 1993; 25: 1943-1950Crossref PubMed Scopus (20) Google Scholar, 18Garrik L.M. Sloan R.L. Thomas W.R. Thomas J.K. Garrik M.D. Biochem. J. 1978; 173: 321-330Crossref PubMed Scopus (46) Google Scholar, 19Chua C.G. Carrel W.R. Howard H.B. Biochem. J. 1975; 170: 259-279Crossref Scopus (40) Google Scholar). In the present study, the reactivities of cysteinyl residues in rat hemoglobin components have been characterized, and the differences have been interpreted in terms of molecular structure. The reported results are consistent with a major role played, in the transient adaptation of rat erythrocyte to oxidative stress, by thiols that do not show conformational-associated changes in their microenvironment.DISCUSSIONSince amino groups generally remain protonated below pH 10, the major nucleophilic functionality available in biological systems are thiols, which, accordingly, are the chemical groups that provide strong defense against electrophilic species. One of the objectives of the present research was to characterize in kinetic terms the reactivity of rat hemoglobin thiols under conditions mimicking the physiological ones. Therefore, this study was limited to free reactive thiols, whereas the so-called masked sulfhydryls, i.e.Cys-104α(G11) and Cys-111α(G18), which are located at the α1β1 interface and whose reactivity is controlled by the tetramer dissociation beyond the dimer stage (52Chiancone E. Currell D.L. Vecchini P. Antonini E. Wyman J. J. Biol. Chem. 1970; 245: 4105-4111Abstract Full Text PDF PubMed Google Scholar), were neglected.The peculiar reactivity of fHbSH present on most of the rat hemoglobin components is clearly responsible for the formation of HbSSG after oxidative stress by diamide. Thiols in proteins are integral to a number of cellular functions, including protein folding, enzyme catalysis, and metabolic regulation (36Creighton T.E. Methods Enzymol. 1984; 107: 305-329Crossref PubMed Scopus (174) Google Scholar, 53Packer L. Methods Enzymol. 1995; 252: XIIICrossref Google Scholar). The reported findings clearly suggest an involvement of fHbSH as a direct moderator of oxidative stress at least in rat erythrocytes. Red cells are regularly subjected to high oxygen tension and are among the first body cells exposed to exogenous oxidative substances that are ingested, inhaled, or injected. The importance of an antioxidant function in erythrocytes is well known and is shown, e.g. by the massive oxidant-induced hemolysis seen in subjects with a marked deficiency of glucose-6-phosphate dehydrogenase (54Luzzatto L. Cell Biochem. Funct. 1987; 5: 101-107Crossref PubMed Scopus (9) Google Scholar).The possible contribution of fHbSH to the intracellular detoxifying mechanism appears to be in line with the definition of the term antioxidant (55Halliwell B. Gutteridge J.M.C. Free Radical Biology and Medicine. Clarendon Press, London1989: 9Google Scholar, 56Sies H. Eur. J. Biochem. 1993; 215: 213-219Crossref PubMed Scopus (1592) Google Scholar) for both the intrinsic chemical properties and the intraerythrocyte concentration. However, this thiol system seems to be suited to resist acute episodes of oxidant fluxes but not the prolonged ones, because its efficiency is somewhat impaired by the long recovery time (see Table I). Since the reactivity of Cys-125β(H3) is not linked to the oxygenation nor to the quaternary state of hemoglobin (see Table III), fHbSH are expected to quickly produceS-conjugates in both the arterial and venous blood; in other words, flow in the blood circulation is expected not to induce a cycle (speeding up and slowing down) in the reactivity of fHbSH, as described for the highly conserved Cys-93β(F9) (and possibly true also for Cys-13α(A11)) that, being oxygen-linked, modulates arterial-venous differences in intraerythrocyte S-nitrosothiols (57Jia L. Bonaventura C. Bonaventura J. Stamler J.S. Nature. 1996; 380: 221-226Crossref PubMed Scopus (1459) Google Scholar, 58Di Simplicio P. Lusini L. Giannerini F. Giustarini D. Bellelli A. Boumis G. Amiconi G. Rossi R. Moncada S. Higgs H.A. Bagetta G. Nitric Oxide and Cell: Proliferation and Death. Portland Press Ltd., London1998Google Scholar). Moreover, the S-conjugation capacity of fHbSH is very high. In fact, from the mean red cell volume (61.6 fl), the mean erythrocyte hemoglobin concentration (21.1 pg), the molecular mass of tetrameric hemoglobin (65 kDa), the fraction of isoforms carrying fHbSH (0.76), and the number of fast reacting cysteinyl residues per tetramer (two), an intracellular level of fHbSH equal to 8 mm can be calculated; this value, which is four times that of glutathione (2 mm), is great enough to force the conclusion that fHbSH works as a buffer system able to trap a large amount of attacking (electrophilic) species in a very short time. Finally, in the particular case investigated it can be also speculated that the efficient nonenzymatic antioxidant defense offered by fHbSH is able to compensate for the poor catalytic activity of glutathione reductase in rat erythrocytes (see Table III).In conclusion and from a more general point of view, the reported results emphasize an additional function of some kinds of protein cysteinyl residues, i.e. a direct detoxification role by conjugation more efficient than low molecular weight thiols, such as glutathione. Human but not rat erythrocytes are reported (1Kosower N.S. Kosower E.M. Dolphin D. Poulson R. Avramovic O. Coenzymes and Cofactors. Vol. III. John Wiley & Sons, Inc., New York1989: 319-356Google Scholar) to be able to restore the cellular pool of GSH, which is strongly decreased after treatment with diazenedicarboxylic acid bis(N,N-dimethylamide), a thiol-oxidizing agent known by the trivial name diamide (2Kosower N.S. Kosower E.M. Methods Enzymol. 1995; 251: 123-133Crossref PubMed Scopus (284) Google Scholar). Such a difference in redox behavior was attributed to a lower enzymatic capacity in reducing disulfides of rat red cells relative to the human ones (3Kosower N.S. Kosower E.M. Koppel R.L. Eur. J. Biochem. 1977; 77: 529-534Crossref PubMed Scopus (42) Google Scholar). However, recent work in this laboratory demonstrated that the reversibility of such process can be observed also in rat erythrocytes, depending on diamide dose (4Di Simplicio P. Lupis E. Rossi R. Biochim. Biophys. Acta. 1996; 1289: 257-275Google Scholar). Additional evidence (3Kosower N.S. Kosower E.M. Koppel R.L. Eur. J. Biochem. 1977; 77: 529-534Crossref PubMed Scopus (42) Google Scholar) may suggest that these differences in behavior between rat and human erythrocytes could be related to diversities in reactivity of sulfhydryl groups of the corresponding hemoglobins. Studies of the reactivities of the sulfhydryl groups of oxygen carriers (5Garel M.C. Beuzard Y. Thillet J. Domenget C. Martin J. Galacteros F. Rosa J. Eur. J. Biochem. 1982; 123: 513-519Crossref PubMed Scopus (47) Google Scholar, 6Taylor J.F. Antonini E. Brunori M. Wyman J. J. Biol. Chem. 1966; 241: 241-248Abstract Full Text PDF PubMed Google Scholar, 7Birchmeier W. Tuchschmid P.E. Winterhalter K. Biochemistry. 1973; 12: 3667-3672Crossref PubMed Scopus (22) Google Scholar, 8Garel M.C. Domenget C. Caburi-Martin J. Prehu C. Galacteros F. Beuzard Y. J. Biol. Chem. 1986; 261: 14704-14709Abstract Full Text PDF PubMed Google Scholar) are mainly restricted to hemoglobins containing only two accessible thiols per tetrameric molecule (such as human hemoglobin), located at the (F9)93 position of each β subunit. Many sulfhydryl reagents can be bound to Cys-93β(F9), even in intact red blood cells (8Garel M.C. Domenget C. Caburi-Martin J. Prehu C. Galacteros F. Beuzard Y. J. Biol. Chem. 1986; 261: 14704-14709Abstract Full Text PDF PubMed Google Scholar). Nevertheless, despite its high intracellular level (about half the concentration of the hemoglobin tetramer (9Beutler E. Red Cell Metabolism: A Manual of Biochemical Methods. 2nd Ed. Grune and Stratton, Inc., Orlando, FL1975: 105-107Google Scholar)), glutathione is not significantly combined with this protein-bound thiol under normal conditions. GSSG in fact is reported (5Garel M.C. Beuzard Y. Thillet J. Domenget C. Martin J. Galacteros F. Rosa J. Eur. J. Biochem. 1982; 123: 513-519Crossref PubMed Scopus (47) Google Scholar, 6Taylor J.F. Antonini E. Brunori M. Wyman J. J. Biol. Chem. 1966; 241: 241-248Abstract Full Text PDF PubMed Google Scholar, 7Birchmeier W. Tuchschmid P.E. Winterhalter K. Biochemistry. 1973; 12: 3667-3672Crossref PubMed Scopus (22) Google Scholar, 8Garel M.C. Domenget C. Caburi-Martin J. Prehu C. Galacteros F. Beuzard Y. J. Biol. Chem. 1986; 261: 14704-14709Abstract Full Text PDF PubMed Google Scholar) (i) to react very slowly with human hemoglobin in vitro and (ii) to spontaneously form with this protein adduct in vivo only under very special conditions and in small amounts (2–7% of the total hemoglobin), having been observed to date solely in hemolysates of patients on long term anti-epileptic therapy (10Niketic V. Beslo D. Raicevic S. Sredic S. Tojkovic M. Int. J. Biochem. 1992; 24: 503-507Crossref PubMed Scopus (10) Google Scholar). On the contrary, evidence exists that rat erythrocytes produce large quantities of glutathione-protein mixed disulfides under any oxidative stress (4Di Simplicio P. Lupis E. Rossi R. Biochim. Biophys. Acta. 1996; 1289: 257-275Google Scholar,11Di Simplicio P. Rossi R. Biochim. Biophys. Acta. 1994; 1199: 245-252Crossref PubMed Scopus (29) Google Scholar, 12Terada T. Nishimura M. Oshida H. Oshida T. Mizoguchi T. Biochem. Mol. Biol. Int. 1993; 29: 1009-1014PubMed Google Scholar, 13Grossman S.J. Jollow D.J. J. Pharmacol. Exp. Ther. 1987; 244: 118-125Google Scholar), most of them being adducts with hemoglobin thiols (HbSSG). 1The abbreviations used are: HbSSG, glutathione-hemoglobin mixed disulfide; t-BOOH,tert-butylhydroperoxide; DTNB, 5,5-dithio-bis(2-nitrobenzoic acid); HbSSHb, hemoglobin-hemoglobin disulfides; HbSH, reactive sulfhydryl groups on hemoglobin; fHbSH, fast reacting sulfhydryl groups on rat hemoglobin components; vsHbSH, slow reacting sulfhydryl groups on rat hemoglobin components; vsHbSH, very slow reacting sulfhydryl groups on rat hemoglobin components; (v)sHbSH, very slow reacting and/or slow reacting sulfhydryl groups on rat hemoglobin components; HPLC, high pressure liquid chromatography. 1The abbreviations used are: HbSSG, glutathione-hemoglobin mixed disulfide; t-BOOH,tert-butylhydroperoxide; DTNB, 5,5-dithio-bis(2-nitrobenzoic acid); HbSSHb, hemoglobin-hemoglobin disulfides; HbSH, reactive sulfhydryl groups on hemoglobin; fHbSH, fast reacting sulfhydryl groups on rat hemoglobin components; vsHbSH, slow reacting sulfhydryl groups on rat hemoglobin components; vsHbSH, very slow reacting sulfhydryl groups on rat hemoglobin components; (v)sHbSH, very slow reacting and/or slow reacting sulfhydryl groups on rat hemoglobin components; HPLC, high pressure liquid chromatography. In particular, treatment of rat red cells with t-BOOH leads to GSH oxidation with formation of both GSSG and HbSSG, followed by a recovery of the initial values within 60 min; on the other hand, addition of diamide to the same system only causes an increase in HbSSG with no formation of GSSG (4Di Simplicio P. Lupis E. Rossi R. Biochim. Biophys. Acta. 1996; 1289: 257-275Google Scholar). All these facts, as a whole, prompted us to investigate further some structural and functional properties of rat hemoglobin thiols. Many hemoglobin components (6Taylor J.F. Antonini E. Brunori M. Wyman J. J. Biol. Chem. 1966; 241: 241-248Abstract Full Text PDF PubMed Google Scholar, 7Birchmeier W. Tuchschmid P.E. Winterhalter K. Biochemistry. 1973; 12: 3667-3672Crossref PubMed Scopus (22) Google Scholar, 8Garel M.C. Domenget C. Caburi-Martin J. Prehu C. Galacteros F. Beuzard Y. J. Biol. Chem. 1986; 261: 14704-14709Abstract Full Text PDF PubMed Google Scholar, 9Beutler E. Red Cell Metabolism: A Manual of Biochemical Methods. 2nd Ed. Grune and Stratton, Inc., Orlando, FL1975: 105-107Google Scholar, 10Niketic V. Beslo D. Raicevic S. Sredic S. Tojkovic M. Int. J. Biochem. 1992; 24: 503-507Crossref PubMed Scopus (10) Google Scholar) have been separated from hemolysates of adult rats (14Stein S. Cherian J. Mazur A. J. Biol. Chem. 1971; 246: 5287-5293Abstract Full Text PDF PubMed Google Scholar, 15Garrik L.M. Sharma S.V. McDonald M.J. Ranney H.M. Biochem. J. 1975; 149: 245-258Crossref PubMed Scopus (65) Google Scholar, 16Datta M.C. Gilman J.G. Hemoglobin. 1981; 5: 701-714Crossref PubMed Scopus (22) Google Scholar), and two types of α subunits and four types of β subunits have been identified (17Ferranti P. Carbone V. Sannolo N. Fiume I. Malorni A. Int. J. Biochem. 1993; 25: 1943-1950Crossref PubMed Scopus (20) Google Scholar). The primary structures reveal the presence of 3 cysteinyl residues on the major α chain (All(13), G11(104), and G18(111)) and up to 2 on the β chain (F9(93) common to all 4 β subunits, and H3(125) in 3 out of 4 β subunits) (15Garrik L.M. Sharma S.V. McDonald M.J. Ranney H.M. Biochem. J. 1975; 149: 245-258Crossref PubMed Scopus (65) Google Scholar, 16Datta M.C. Gilman J.G. Hemoglobin. 1981; 5: 701-714Crossref PubMed Scopus (22) Google Scholar, 17Ferranti P. Carbone V. Sannolo N. Fiume I. Malorni A. Int. J. Biochem. 1993; 25: 1943-1950Crossref PubMed Scopus (20) Google Scholar, 18Garrik L.M. Sloan R.L. Thomas W.R. Thomas J.K. Garrik M.D. Biochem. J. 1978; 173: 321-330Crossref PubMed Scopus (46) Google Scholar, 19Chua C.G. Carrel W.R. Howard H.B. Biochem. J. 1975; 170: 259-279Crossref Scopus (40) Google Scholar). In the present study, the reactivities of cysteinyl residues in rat hemoglobin components have been characterized, and the differences have been interpreted in terms of molecular structure. The reported results are consistent with a major role played, in the transient adaptation of rat erythrocyte to oxidative stress, by thiols that do not show conformational-associated changes in their microenvironment. DISCUSSIONSince amino groups generally remain protonated below pH 10, the major nucleophilic functionality available in biological systems are thiols, which, accordingly, are the chemical groups that provide strong defense against electrophilic species. One of the objectives of the present research was to characterize in kinetic terms the reactivity of rat hemoglobin thiols under conditions mimicking the physiological ones. Therefore, this study was limited to free reactive thiols, whereas the so-called masked sulfhydryls, i.e.Cys-104α(G11) and Cys-111α(G18), which are located at the α1β1 interface and whose reactivity is controlled by the tetramer dissociation beyond the dimer stage (52Chiancone E. Currell D.L. Vecchini P. Antonini E. Wyman J. J. Biol. Chem. 1970; 245: 4105-4111Abstract Full Text PDF PubMed Google Scholar), were neglected.The peculiar reactivity of fHbSH present on most of the rat hemoglobin components is clearly responsible for the formation of HbSSG after oxidative stress by diamide. Thiols in proteins are integral to a number of cellular functions, including protein folding, enzyme catalysis, and metabolic regulation (36Creighton T.E. Methods Enzymol. 1984; 107: 305-329Crossref PubMed Scopus (174) Google Scholar, 53Packer L. Methods Enzymol. 1995; 252: XIIICrossref Google Scholar). The reported findings clearly suggest an involvement of fHbSH as a direct moderator of oxidative stress at least in rat erythrocytes. Red cells are regularly subjected to high oxygen tension and are among the first body cells exposed to exogenous oxidative substances that are ingested, inhaled, or injected. The importance of an antioxidant function in erythrocytes is well known and is shown, e.g. by the massive oxidant-induced hemolysis seen in subjects with a marked deficiency of glucose-6-phosphate dehydrogenase (54Luzzatto L. Cell Biochem. Funct. 1987; 5: 101-107Crossref PubMed Scopus (9) Google Scholar).The possible contribution of fHbSH to the intracellular detoxifying mechanism appears to be in line with the definition of the term antioxidant (55Halliwell B. Gutteridge J.M.C. Free Radical Biology and Medicine. Clarendon Press, London1989: 9Google Scholar, 56Sies H. Eur. J. Biochem. 1993; 215: 213-219Crossref PubMed Scopus (1592) Google Scholar) for both the intrinsic chemical properties and the intraerythrocyte concentration. However, this thiol system seems to be suited to resist acute episodes of oxidant fluxes but not the prolonged ones, because its efficiency is somewhat impaired by the long recovery time (see Table I). Since the reactivity of Cys-125β(H3) is not linked to the oxygenation nor to the quaternary state of hemoglobin (see Table III), fHbSH are expected to quickly produceS-conjugates in both the arterial and venous blood; in other words, flow in the blood circulation is expected not to induce a cycle (speeding up and slowing down) in the reactivity of fHbSH, as described for the highly conserved Cys-93β(F9) (and possibly true also for Cys-13α(A11)) that, being oxygen-linked, modulates arterial-venous differences in intraerythrocyte S-nitrosothiols (57Jia L. Bonaventura C. Bonaventura J. Stamler J.S. Nature. 1996; 380: 221-226Crossref PubMed Scopus (1459) Google Scholar, 58Di Simplicio P. Lusini L. Giannerini F. Giustarini D. Bellelli A. Boumis G. Amiconi G. Rossi R. Moncada S. Higgs H.A. Bagetta G. Nitric Oxide and Cell: Proliferation and Death. Portland Press Ltd., London1998Google Scholar). Moreover, the S-conjugation capacity of fHbSH is very high. In fact, from the mean red cell volume (61.6 fl), the mean erythrocyte hemoglobin concentration (21.1 pg), the molecular mass of tetrameric hemoglobin (65 kDa), the fraction of isoforms carrying fHbSH (0.76), and the number of fast reacting cysteinyl residues per tetramer (two), an intracellular level of fHbSH equal to 8 mm can be calculated; this value, which is four times that of glutathione (2 mm), is great enough to force the conclusion that fHbSH works as a buffer system able to trap a large amount of attacking (electrophilic) species in a very short time. Finally, in the particular case investigated it can be also speculated that the efficient nonenzymatic antioxidant defense offered by fHbSH is able to compensate for the poor catalytic activity of glutathione reductase in rat erythrocytes (see Table III).In conclusion and from a more general point of view, the reported results emphasize an additional function of some kinds of protein cysteinyl residues, i.e. a direct detoxification role by conjugation more efficient than low molecular weight thiols, such as glutathione. Since amino groups generally remain protonated below pH 10, the major nucleophilic functionality available in biological systems are thiols, which, accordingly, are the chemical groups that provide strong defense against electrophilic species. One of the objectives of the present research was to characterize in kinetic terms the reactivity of rat hemoglobin thiols under conditions mimicking the physiological ones. Therefore, this study was limited to free reactive thiols, whereas the so-called masked sulfhydryls, i.e.Cys-104α(G11) and Cys-111α(G18), which are located at the α1β1 interface and whose reactivity is controlled by the tetramer dissociation beyond the dimer stage (52Chiancone E. Currell D.L. Vecchini P. Antonini E. Wyman J. J. Biol. Chem. 1970; 245: 4105-4111Abstract Full Text PDF PubMed Google Scholar), were neglected. The peculiar reactivity of fHbSH present on most of the rat hemoglobin components is clearly responsible for the formation of HbSSG after oxidative stress by diamide. Thiols in proteins are integral to a number of cellular functions, including protein folding, enzyme catalysis, and metabolic regulation (36Creighton T.E. Methods Enzymol. 1984; 107: 305-329Crossref PubMed Scopus (174) Google Scholar, 53Packer L. Methods Enzymol. 1995; 252: XIIICrossref Google Scholar). The reported findings clearly suggest an involvement of fHbSH as a direct moderator of oxidative stress at least in rat erythrocytes. Red cells are regularly subjected to high oxygen tension and are among the first body cells exposed to exogenous oxidative substances that are ingested, inhaled, or injected. The importance of an antioxidant function in erythrocytes is well known and is shown, e.g. by the massive oxidant-induced hemolysis seen in subjects with a marked deficiency of glucose-6-phosphate dehydrogenase (54Luzzatto L. Cell Biochem. Funct. 1987; 5: 101-107Crossref PubMed Scopus (9) Google Scholar). The possible contribution of fHbSH to the intracellular detoxifying mechanism appears to be in line with the definition of the term antioxidant (55Halliwell B. Gutteridge J.M.C. Free Radical Biology and Medicine. Clarendon Press, London1989: 9Google Scholar, 56Sies H. Eur. J. Biochem. 1993; 215: 213-219Crossref PubMed Scopus (1592) Google Scholar) for both the intrinsic chemical properties and the intraerythrocyte concentration. However, this thiol system seems to be suited to resist acute episodes of oxidant fluxes but not the prolonged ones, because its efficiency is somewhat impaired by the long recovery time (see Table I). Since the reactivity of Cys-125β(H3) is not linked to the oxygenation nor to the quaternary state of hemoglobin (see Table III), fHbSH are expected to quickly produceS-conjugates in both the arterial and venous blood; in other words, flow in the blood circulation is expected not to induce a cycle (speeding up and slowing down) in the reactivity of fHbSH, as described for the highly conserved Cys-93β(F9) (and possibly true also for Cys-13α(A11)) that, being oxygen-linked, modulates arterial-venous differences in intraerythrocyte S-nitrosothiols (57Jia L. Bonaventura C. Bonaventura J. Stamler J.S. Nature. 1996; 380: 221-226Crossref PubMed Scopus (1459) Google Scholar, 58Di Simplicio P. Lusini L. Giannerini F. Giustarini D. Bellelli A. Boumis G. Amiconi G. Rossi R. Moncada S. Higgs H.A. Bagetta G. Nitric Oxide and Cell: Proliferation and Death. Portland Press Ltd., London1998Google Scholar). Moreover, the S-conjugation capacity of fHbSH is very high. In fact, from the mean red cell volume (61.6 fl), the mean erythrocyte hemoglobin concentration (21.1 pg), the molecular mass of tetrameric hemoglobin (65 kDa), the fraction of isoforms carrying fHbSH (0.76), and the number of fast reacting cysteinyl residues per tetramer (two), an intracellular level of fHbSH equal to 8 mm can be calculated; this value, which is four times that of glutathione (2 mm), is great enough to force the conclusion that fHbSH works as a buffer system able to trap a large amount of attacking (electrophilic) species in a very short time. Finally, in the particular case investigated it can be also speculated that the efficient nonenzymatic antioxidant defense offered by fHbSH is able to compensate for the poor catalytic activity of glutathione reductase in rat erythrocytes (see Table III). In conclusion and from a more general point of view, the reported results emphasize an additional function of some kinds of protein cysteinyl residues, i.e. a direct detoxification role by conjugation more efficient than low molecular weight thiols, such as glutathione." @default.
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• W2016553301 title "Fast-reacting Thiols in Rat Hemoglobins Can Intercept Damaging Species in Erythrocytes More Efficiently Than Glutathione" @default.
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