Hemoglobin polymerization via disulfide bond formation in the hypoxia-tolerant turtleTrachemys scripta: implications for antioxidant defense and O₂transport

Abstract

The ability of many reptilian hemoglobins (Hbs) to form high-molecular weight polymers, albeit known for decades, has not been investigated in detail. Given that turtle Hbs often contain a high number of cysteine (Cys), potentially contributing to the red blood cell defense against reactive oxygen species, we have examined whether polymerization of Hb could occur via intermolecular disulfide bonds in red blood cells of freshwater turtle Trachemys scripta, a species that is highly tolerant of hypoxia and oxidative stress. We find that one of the two Hb isoforms of the hemolysate HbA is prone to polymerization in vitro into linear flexible chains of different size that are visible by electron microscopy but not the HbD isoform. Polymerization of purified HbA is favored by hydrogen peroxide, a main cellular reactive oxygen species and a thiol oxidant, and inhibited by thiol reduction and alkylation, indicating that HbA polymerization is due to disulfide bonds. By using mass spectrometry, we identify Cys5 of the α-subunit of HbA as specifically responsible for forming disulfide bonds between adjacent HbA tetramers. Polymerization of HbA does not affect oxygen affinity, cooperativity, and sensitivity to the allosteric cofactor ATP, indicating that HbA is still fully functional. Polymers also form in T. scripta blood after exposure to anoxia but not normoxia, indicating that they are of physiological relevance. Taken together, these results show that HbA polymers may form during oxidative stress and that Cys5α of HbA is a key element of the antioxidant capacity of turtle red blood cells.

Tissue-dependent variations of hydrogen sulfide homeostasis in anoxic freshwater turtles

“…2B). These high values of BSS in RBCs appear to be consistent with the high content (∼24 mmol l −1 ) of total reactive thiols in T. scripta elegans RBCs, including those of hemoglobin (Hb) and GSH (Damsgaard et al, 2013;Jacobsen et al, 2012;Petersen et al, 2018). Thiols can potentially be partly sulfhydrated (i.e.…”

“…Thiols can potentially be partly sulfhydrated (i.e. R-S-SH) and contribute to the BSS pool, and in T. scripta elegans Hb, we have previously identified bound persulfide and polysulfides by mass spectrometry (Petersen et al, 2018). Considering that protein sulfhydration is a pervasive modification (Mishanina et al, 2015) estimated to be up to 10-25% for certain proteins (Mustafa et al, 2009), the values of ∼70-120 µmol l −1 detected here indicate a maximum of 0.25-0.5% of total protein thiol sulfhydration in turtle RBCs, which is not unrealistic.…”

Regulatory Variation in Functionally Polymorphic Globin Genes of the Bank Vole: A Possible Role for Adaptation

“…Similar to reactive cysteine-containing Hbs of other rodents (Rossi et al, 1998;Storz et al, 2012), bank vole isoform incorporating the β-globin chain encoded by the Cys52 allele (Hb F) significantly increases the resistance of RBCs to oxidative stress (Kotlík et al, 2014). This resistant phenotype is explained by Hb F taking part in the RBC protein redox system involved in ROS detoxification via the highly-reactive thiol (-SH) group of Cys52, together with the glutathione (GSH) redox system (Rossi et al, 1998;Kotlík et al, 2014;Petersen et al, 2017). Because of the very high concentration of Hb in RBCs, regulatory variation differentially affecting the contribution of the β-globin alleles that differ in Cys content to Hb could provide an effective mechanism for modulating the antioxidant capacity of RBCs (Rossi et al, 1998;Storz et al, 2012;Petersen et al, 2017).…”

“…A recent study showed that Hb F homozygotes have significantly higher antioxidant capacity of the red blood cells (RBCs) than Hb S homozygotes, which is due to a single amino acid substitution of serine (Ser) with cysteine (Cys) at position 52 in the β subunit of Hb F (Kotlík et al, 2014). The explanation is that the thiol (-SH) group of the side chain of Cys52 in Hb F takes part in the RBC protein redox system involved in detoxification of reactive oxygen species (ROS) (Rossi et al, 1998;Storz et al, 2012;Kotlík et al, 2014;Petersen et al, 2017). Previous studies found evidence suggesting that the resistant RBC phenotype is advantageous under environmental conditions conducive of oxidative stress (Kotlík et al, 2014;Strážnická et al, 2018).…”

New insights into the allosteric effects of CO2and bicarbonate on crocodilian hemoglobin

“…2C) and non-reducing conditions (Fig. S3) showed identical patterns and intensity of bands indicating that high-order aggregates are not formed via disulfide bonds as in the Hb aggregates of other crocodilians (Weber et al, 2013) and non-avian reptiles (Petersen et al, 2017; Storz et al, 2015).…”