Binding of 4‐hydroxy‐2‐nonenal Decrease Non‐enzymatic Methemoglobin Reducing Capacity

Abstract

Lipid peroxidation can produce reactive primary and secondary oxidation products. Various studies have shown that secondary lipid oxidation products, such as 4‐hydroxy‐2‐nonenal (HNE), can bind with proteins and minimize their functional properties. Hemoglobin is the primary oxygen‐carrying protein and can exist in three different forms; oxyhemoglobin, deoxyhemoglobin, and methemoglobin. Oxyhemoglobin/deoxyhemoglobin can carry oxygen, while the oxidized form, methemoglobin, cannot. Physiologically, methemoglobin can be converted to deoxyhemoglobin by enzymatic and non‐enzymatic reduction. Studies have shown that HNE can preferentially bind with histidine and lysine residues within a protein by Michael addition; Hemoglobin has several histidine and lysine residues in alpha and beta structures. However, limited studies have determined the effects of HEN binding on non‐enzymatic methemoglobin reducing capacity. The overall goal of this study was to determine the effects of HNE on non‐enzymatic methemoglobin reducing capacity. Equine hemoglobin (0.1 mM) was pre‐incubated with HNE (0, 0.1, and 0.7 mM) at pH 7.4 and 37 °C for 5 hours to promote covalent binding of HNE to methemoglobin. Following pre‐incubation, methemoglobin was combined with methylene blue and EDTA. The non‐enzymatic reduction was initiated by the addition of NADH and the reduction of methemoglobin was monitored using a UV‐Vis spectrophotometer. Methemoglobin reduction was expressed as nanomoles of methemoglobin reduced per min. The experiment was replicated three times. Pre‐incubation of HNE decreased (p < 0.05) non‐enzymatic methemoglobin reducing capacity. There were concentration‐dependent effects of HNE on non‐enzymatic reduction. Methemoglobin reduction was decreased (p < 0.05) by 17% for 0.1 mM HNE compared with control methemoglobin, however, there was 60% decrease (p < 0.05) in non‐enzymatic methemoglobin reducing activity with 0.7 mM HNE. Lipid peroxidation in various physiological and pathological conditions can increase HNE level in tissue and plasma. The current study suggests that HNE can decrease non‐enzymatic hemoglobin reduction.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.