Abstract
Hemoglobin is the natural carrier of oxygen in red blood cells (RBCs). While intracellular hemoglobin provides life-sustaining oxygen transport, extracellular free hemoglobin displays toxicity due to inherent peroxidase activity generating reactive oxygen species that subsequently react with the hemoglobin molecule to produce toxic heme degradation products resulting in free radicals, oxidative stress damage, and lipid peroxidation. We have recently demonstrated that Peptide Inhibitor of Complement C1 (PIC1) inhibits peroxidase activity of the heme-based enzyme myeloperoxidase. To elucidate whether PIC1 could inhibit peroxidase activity of hemoglobin, we evaluated the consequence of PIC1 on RBC lysates, methemoglobin, and myoglobin using tetramethylbenzidine (TMB) as an oxidation target. PIC1 reversibly and dose-dependently prevented TMB oxidation to tetramethylbenzidine diimine by RBC lysates, methemoglobin, and myoglobin, having comparable activity to the inhibitor 4-aminobenzoic acid hydrazide. PIC1 inhibited TMB oxidation of RBC lysates similar to L-cysteine suggesting that the two cysteine residues contained in PIC1 may mediate peroxidase activity. PIC1 also inhibited heme destruction by NaOCl for RBC lysates, hemoglobin, and myoglobin as assayed by preservation of the Soret absorbance peak in the presence of NaOCl and reduction in free iron release. In conclusion, PIC1 inhibits peroxidase activity of hemoglobin and myoglobin likely via an antioxidant mechanism.
Highlights
The hemoglobin (Hb) molecule is a protein tetramer composed of two alpha and two beta chains each of which coordinates an iron-containing heme prosthetic group [1]
In an attempt to ascertain whether Peptide Inhibitor of Complement C1 (PIC1) would have any effect on the peroxidase activity of extracellular Hb, red blood cells (RBCs) lysates were prepared from four donors and incubated with increasing amounts of PIC1 followed by TMB as the target for oxidation
Addition of PIC1 led to a dose-dependent inhibition of the TMB oxidation product 3,3,5,5-tetramethylbenzidine diimine (Figure 1(a)) demonstrating a 24.8-fold reduction in peroxidase activity for 7.5 mM PIC1 compared with no PIC1 (P < 0.001)
Summary
The hemoglobin (Hb) molecule is a protein tetramer composed of two alpha and two beta chains each of which coordinates an iron-containing heme prosthetic group [1]. While Hb provides life-sustaining oxygen delivery, the Hb molecule is inherently toxic due to its well-characterized redox activity leading to autoxidation of the ferrousHb (Fe2+) oxygen containing Hb (oxyHb) into nonfunctional ferricHb (Fe3+) known as methemoglobin (metHb) and reactive oxygen species (ROS) generation of superoxide ion (O2∙−) [2]. Within the confines of the RBC, autoxidation is minimized by the metHb reductase system; free metHb can undergo a further peroxidase reaction with O2∙− to generate ferrylHb (Fe4+) and the ROS hydrogen peroxide (H2O2) that in turn attacks Hb to produce protein radicals, irreversibly damaging the Hb molecule leading to heme degradation and release of free iron [2]. In addition to generation of ROS from free Hb, iron released from heme degradation can result in International Journal of Peptides the generation of additional ROS resulting in cellular damage. There are no pharmacological agents available on the market to control pathological extracellular Hb-mediated oxidative reactions
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