Detection and Removal of Endogenous Carbon Monoxide by Selective and Cell-Permeable Hemoprotein Model Complexes

Abstract

Carbon monoxide (CO) is produced in mammalian cells during heme metabolism and serves as an important signaling messenger. Here we report the bioactive properties of selective CO scavengers, hemoCD1 and its derivative R8-hemoCD1, which have the ability to detect and remove endogenous CO in cells. HemoCD1 is a supramolecular hemoprotein-model complex composed of 5,10,15,20-tetrakis(4-sulfonatophenyl)porphinatoiron(II) and a per-O-methylated β-cyclodextrin dimer having an pyridine linker. We demonstrate that hemoCD1 can be used effectively to quantify endogenous CO in cell lysates by a simple spectrophotometric method. The hemoCD1 assay detected ca. 260 pmol of CO in 10 hepatocytes, which was well-correlated with the amount of intracellular bilirubin, the final breakdown product of heme metabolism. We then covalently attached an octaarginine peptide to a maleimide-appended hemoCD1 to synthesize R8-hemoCD1, a cell-permeable CO scavenger. Indeed, R8-hemoCD1 was taken up by intact cells and captured intracellular CO with high efficiency. Moreover, we revealed that removal of endogenous CO by R8-hemoCD1 in cultured macrophages led to a significant increase (ca. 2.5-fold) in reactive oxygen species production and exacerbation of inflammation after challenge with lipopolysaccharide. Thus, R8-hemoCD1 represents a powerful expedient for exploring specific and still unidentified biological functions of CO in cells.

HYCO-3, a dual CO-releaser/Nrf2 activator, reduces tissue inflammation in mice challenged with lipopolysaccharide

“…This could indicate that either HYCO-6 cannot enter the cell and deliver CO or that there is a different chemical reactivity of HYCO-3 and HYCO-6 towards COP-1, the probe we used to detect intracellular CO. Notably, we observed similar results using hemoCD, a unique CO scavenger that binds CO with high affinity and that allows the determination of intracellular CO levels [45] , [46] , confirming that only the manganese-containing HYCO-3, unlike its ruthenium-based counterpart (HYCO-6), delivers CO to cells at the concentrations used (data not shown, manuscript in preparation). This difference may also be explained by the increased bioavailability of HYCO-3, since we found that HYCO-3 is significantly more stable than HYCO-6 in plasma and microsomes.…”

Carbon monoxide–induced metabolic switch in adipocytes improves insulin resistance in obese mice

“…CO released into the vial headspace was quantified by gas chromatography. CO accumulated in cultured 3T3-L1 adipocytes after treatment with CORM-401 was measured spectrophotometrically using a specific scavenger of CO (hemoCD1) as previously described (48).…”

Circadian clock disruption by selective removal of endogenous carbon monoxide

“…To the best of our knowledge, the CO-binding affinity of hemoCD1 in aqueous solutions is the highest among the reported CO-binding hemoproteins. More advantageously, the NO-binding affinity of hemoCD1 is lower than that of hemoglobin 35 . The coordination strength of H 2 S to a hemoCD1 analog is also relatively weak, and the SH – ligand is easily replaceable with CO 36 .…”