The Redox Potential of the β-93-Cysteine Thiol Group in Human Hemoglobin Estimated from In Vitro Oxidant Challenge Experiments

Abstract

Glutathionyl hemoglobin is a minor form of hemoglobin with intriguing properties. The measurement of the redox potential of its reactive β-93-Cysteine is useful to improve understanding of the response of erythrocytes to transient and chronic conditions of oxidative stress, where the level of glutathionyl hemoglobin is increased. An independent literature experiment describes the recovery of human erythrocytes exposed to an oxidant burst by measuring glutathione, glutathione disulfide and glutathionyl hemoglobin in a two-hour period. This article calculates a value for the redox potential E0 of the β-93-Cysteine, considering the erythrocyte as a closed system at equilibrium described by the Nernst equation and using the measurements of the literature experiment. The obtained value of E0 of −121 mV at pH 7.4 places hemoglobin as the most oxidizing thiol of the erythrocyte. By using as synthetic indicators of the concentrations the electrochemical potentials of the two main redox pairs in the erythrocytes, those of glutathione–glutathione disulfide and of glutathionyl–hemoglobin, the mechanism of the recovery phase can be hypothesized. Hemoglobin acts as the redox buffer that scavenges oxidized glutathione in the oxidative phase and releases it in the recovery phase, by acting as the substrate of the NAD(P)H-cofactored enzymes.

Changes in Hemoglobin Properties in Complex with Glutathione and after Glutathionylation

“…Hemoglobin contains six cysteine residues, one each in the alpha subunits (Cys104) and two each in the beta subunits (Cys93 and Cys112). Currently, glutathionylation at Cys93β [35], and Cys112β [28] residues has been proven, while data on Cys-104α glutathionylation are contradictory [3,36].…”

Enhanced-Precision Measurement of Glutathionyl Hemoglobin by MALDI-ToF MS

“…Improved data quality may encourage expanding the use of glutathionyl-hemoglobin as a biomarker of systemic oxidative stress in several human conditions and diseases [ 26 ], to compare it with other biomarkers of the thiol-disulphide metabolome, such as the glutathione-glutathione disulfide redox pair [ 3 , 5 , 27 ]. Accurate and precise measurement of glutathionyl hemoglobin with the fast MALDI-ToF mass spectrometry technique can help solve still poorly understood issues on the different mechanisms of its generation.…”

“…Vice-versa, the disulfide bond can be reversibly cleaved in a trans-sulfuration reaction [ 3 ] with a nucleophilic free thiol group that belongs to a compound with a lower redox potential [ 4 , 5 ]. These good reducing agent are in-vivo glutathione (pathway a ), drugs such as N -acetyl-cysteine and in-vitro glutathione, N -acetyl-cysteine and biochemical tools, such as dithio-treitol.…”

Differential Redox State and Iron Regulation in Chronic Obstructive Pulmonary Disease, Acute Respiratory Distress Syndrome and Coronavirus Disease 2019

“…However, the glutathione units are in part bound to Hb to form glutathionyl Hb through a disulfide bond to the thiol group of the β93 cysteine residue. Due to its peculiar electrochemical redox potential, this minor form of Hb has been predicted to act as a redox buffer that scavenges oxidized glutathione in the oxidative phase and releases it in the recovery phase [ 46 ]. Remarkably, the level of glutathione bound to Hb is higher in COPD than it is in the controls, which re-establishes the size of the glutathione pool in chronically hypoxic patients.…”