Cysteine 93 of Hemoglobin Beta Chain Is the Major Target of Oxidation During Red Blood Cell Storage.
Authors: Blacken Grady R. , Wang Yi , Lopez Jose A. , Fu Xiaoyun 
Published: October 2019

Abstract 4040

Poster Board III-976

Red blood cells (RBC) stored for prolonged time periods under blood banking conditions are known to accumulate irreversible damage that ultimately affects their viability and function. Among the consequences of this RBC storage lesion are enhanced hemolysis and oxidative stress after transfusion. Stored RBCs accumulate reactive oxygen species (ROS) and surface-associated hemichrome, a partially denatured form of oxidized hemoglobin. In this work, we examined whether the ROS created during RBC storage lead to characteristic oxidative modifications to hemoglobin (Hb) and RBC membrane proteins.

To characterize Hb modifications leading to hemichrome formation, we oxidized human hemoglobin A with varied concentrations of hydrogen peroxide (H2O2), an oxidant relevant to RBC storage. The reaction was quenched with excess methionine and the protein digested with trypsin. The resulting peptides were analyzed by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS). Methionine and cysteine residues are the major targets of oxidation. Oxidation of methionine to methionine sulfoxide and cysteine to cysteine sulfonic/sulfinic acids is accompanied by a mass shift related to the covalent modifications of each residue, which are readily detected by LC-ESI-MS/MS. We found that HBβ-Cys-93 was oxidized to a maximum of approximately 70% after treatment with 200 αM H2O2. This cysteine occurs adjacent to the FeII-coordinating His-92 and is thus exposed to the heme-FeII center. In contrast, HBβ-Cys-112 and HBα-Cys-104, which are buried and located away from the heme-FeII center, were oxidized to only 2% and 0%, respectively. The enhanced oxidation of the heme-FeII proximal HBβ-Cys-93 suggests that ROS are generated through a FeII-dependent mechanism such as Fenton's reaction. Oxidation of each of the three Hb methionines reached 20% upon treatment with 200 μM H2O2. Among these Met residues, HBα-Met-32 is located near the heme-FeII, center, while both Hbα-Met-76 and HBβ-Met-55 are near the protein/solvent interface. Thus, Cys and Met oxidations in Hb appear to be dependent on proximity to either the heme-FeII center or the protein/solvent interface.

To test whether these Hb residues are also sensitive to oxidation during storage we acquired 3 RBC units stored for 3 days and 3 RBC units stored for 45 days, from the Puget Sound Blood Center. The cytosolic Hb was extracted by hypotonic lysis and digested with trypsin prior to LC-ESI-MS/MS analysis. We observed that oxidation of HBβ-Cys-93 was dramatically increased from 10% on day 3 to 29% on day 45 (p < 0.02). HBα-Met-32 was also oxidized to a small extent (5% on day 3 to 7.5 % on day 45, p<0.04). The two buried Cys residues were unoxidized, whereas two surface proximal Met residues were oxidized, but not in a significantly storage-time-dependent manner. These data implicate the redox active heme-FeII center in enhanced ROS production and oxidative stress in stored RBCs.

Our observations suggest that HBβ-Cys-93 oxidation is a specific and sensitive marker for oxidative stress during storage. Moreover, we have correlated these progressive oxidations with increases in microvesiculation and hemolysis, storage-time-dependent changes that have been well documented. Future studies will evaluate the storage-time-dependence in other oxidative and post-translational modifications to RBC membrane proteins and correlate these findings with functional consequences such as microvesiculation and hemolysis.

Disclosures:

No relevant conflicts of interest to declare.