Thiol groups in proteins as endogenous reductants to determine glutathione-protein mixed disulphides in biological systems

Rossi, Ranieri; Cardaioli, Elena; Scaloni, Andrea; Amiconi, Gino; Simplicio, P. Di

doi:10.1016/0304-4165(94)00133-i

Cited by 78 publications

(60 citation statements)

References 21 publications

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“…The latter result was in line with the evidence that after treatment of rat hemolysate with dithiothreitol the peak corresponding to the G component disappeared and peak F increased (Fig. 3, panel B) (meaning that isoform G corresponded to the component F with one thiol blocked by glutathione, this reactant being identified by colorimetric and HPLC procedure (22)). …”

Section: S-thiolation Of Hemoglobin Components In Rat Erythrocytessupporting

confidence: 85%

“…Colorimetric determination of GSH was performed by Ellman's method, slightly modified (22), adding DTNB as the last reagent (0.1 mM final concentration, end of reaction at 2 min). GSSG was assayed enzymatically by the procedure of Di Simplicio et al (21).…”

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confidence: 99%

“…Hemoglobin Thiol Titration-Exposed sulfhydryl groups (HbSH) of hemoglobin were assayed by a modification of Ellman's method (22) (conditions, 0.1 M phosphate buffer pH 7.4; DTNB, 0.2 mM final concentration). Monitoring traces were recorded by a stopped-flow apparatus (Applied Photophysics, UK) at room temperature and read at 450 nm; at this wavelength the HbSH concentration was calculated taking ⑀ ϭ 7.0 mM Ϫ1 cm…”

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confidence: 99%

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Fast-reacting Thiols in Rat Hemoglobins Can Intercept Damaging Species in Erythrocytes More Efficiently Than Glutathione

Rossi¹,

Barra²,

Bellelli³

et al. 1998

Journal of Biological Chemistry

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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 t1 ⁄2 <100 ms; slow reacting (sHbSH), with t1 ⁄2 30 -50 s; and very slow reacting (vsHbSH), with t1 ⁄2 180 -270 s. Under paraphysiological conditions, fHbSH (identified with Cys-125␤(H3)) conjugates with DTNB 100 times faster than glutathione and ϳ4000 times more rapidly than (v)sHbSH (Cys-13␣(A11) and Cys-93␤(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 (ϳ6.9, the cysteinyl anion being stabilized by a hydrogen bond with Ser-123␤(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 (1) 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 (2). 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 (3). However, recent work in this laboratory demonstrated that the reversibility of such process can be observed also in rat erythrocytes, depending on diamide dose (4). Additional evidence (3) 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.

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Section: S-thiolation Of Hemoglobin Components In Rat Erythrocytessupporting

confidence: 85%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Fast-reacting Thiols in Rat Hemoglobins Can Intercept Damaging Species in Erythrocytes More Efficiently Than Glutathione

Rossi¹,

Barra²,

Bellelli³

et al. 1998

Journal of Biological Chemistry

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“…Осадок использовали для определения содержания смешанных дисульфидов глутатиона с белками (PSSG) по методу, описанному Rossi с соавт. [12]. При определении концентрации GSH в постмитохондриальном супернатанте исключали процедуру замораживания-оттаивания.…”

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Hepatotoxic efects of acetaminophen. Protective properties of tryptophan-derivatives

et al. 2010

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При интоксикации крыс ацетаминофеном (APAP) (500-1500 мг/кг, внутрижелудочно) наблюдали значительное дозозависимое снижение уровня цитоплазматического и внутримитохондриального восстановленного глутатиона (GSH) в ткани печени (при дозе 1500 мг/кг на 60% и 33%, соответственно). В большей степени истощался цитоплазматический GSH по сравнению с митохондриальным. Одновременно, нами не было обнаружено существенной инактивации митохондриальных ферментов: сукцинатдегидрогеназы, a-кетоглутаратдегидрогеназы, глутатионпероксидазы, а также снижения дыхательной активности митохондрий печени крыс, подвергшихся интоксикации APAP, несмотря на значительное исчерпание митохондриального GSH. Мы исследовали при АРАР-индуцируемой интоксикации гепатопротекторные свойства производных триптофана -мелатонина и N-ацетил-N-нитрозотриптофана (NNT) -донора оксида азота. Известный антиоксидант -гормон эпифиза мелатонин (10 мг/кг), существенно не предохраняя внутримитохондриальный GSH, уменьшал степень токсического поражения печени, о чем свидетельствовало снижение активности маркерных ферментов повреждения клеток печени (AлT, AсT) и содержания общего билирубина в плазме крови интоксицированных крыс, в то время как NNT не оказывал гепатопротекторного действия.Ключевые слова: ацетаминофен, мелатонин, тканевое дыхание, окислительный стресс, гепатотоксичность, гепатопротекторы.

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“…tert-Butyl hydroperoxide (BHP) is a commonly used compound to investigate the effects of peroxides and radicals on biological systems. [6][7][8][9] Many lines of evidence have emerged to support a fundamental role of redox modification of certain proteins in normal cellular biochemical and physiological processes. A number of purified proteins are sensitive to oxidation by thiol disulfide interchange reactions.…”

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confidence: 99%

Pyruvate Kinase Is Protected by Glutathione-Dependent Redox Balance in Human Red Blood Cells Exposed to Reactive Oxygen Species

Ogasawara

Funakoshi

Ishii

2008

Biological & Pharmaceutical Bulletin

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To determine the antioxidant role of glutathione (GSH) in human red blood cells (RBCs), we investigated the effect of disrupting GSH homeostasis on the oxidative modification of thiol-dependent enzymes by exposure to tert-butyl hydroperoxide (BHP). When hemolysate was incubated with BHP, significant decreases in enzyme activity were observed. However, the inactivation did not occur in intact RBC suspensions that were exposed to BHP. In this study, we used two independent treatments aimed at decreasing the level of reduced form of GSH, pre-incubation with a glutathione reductase inhibitor or glucose-free medium to examine the influences of preventing GSH-dependent antioxidant and reactivation activity on thiol-dependent enzyme. Pyruvate kinase (PK) activity clearly decreased along with depletion of GSH compared to other glycolytic enzyme activities by BHP exposure in RBCs. The addition of GSH, but not glucose, before BHP exposure completely prevented the inactivation of PK in hemolysate; however, partial reactivation of inactivated PK was observed by post-addition of both GSH and glutaredoxin at an early stage during BHP exposure. Moreover, hydroxyl radicals but not hydrogen peroxide inactivated PK. These results suggest that PK is highly susceptible to radicals and that GSH is essential to protect PK activity by not only directly scavenging radicals but also by systematically reactivating oxidized enzyme in human RBCs.

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Thiol groups in proteins as endogenous reductants to determine glutathione-protein mixed disulphides in biological systems

Cited by 78 publications

References 21 publications

Fast-reacting Thiols in Rat Hemoglobins Can Intercept Damaging Species in Erythrocytes More Efficiently Than Glutathione

Fast-reacting Thiols in Rat Hemoglobins Can Intercept Damaging Species in Erythrocytes More Efficiently Than Glutathione

Hepatotoxic efects of acetaminophen. Protective properties of tryptophan-derivatives

Pyruvate Kinase Is Protected by Glutathione-Dependent Redox Balance in Human Red Blood Cells Exposed to Reactive Oxygen Species

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