By Joe Boyle

Heme oxygenase-1 (HO-1) is a critical determinant of vascular homeostasis, health and disease. HO-1 deficiency causes death from overwhelming vascular disease in childhood. HO-1 degrade heme, which is potentially toxic due to oxidative stress. HO-1 is induced by tissue hemorrhage, which it helps resorb. HO-1 is induced in hemorrhages in atherosclerosis, intracranial hemorrhage, and in glomerulonephritis. An understanding of its role in vasculitis would be an enormous step forwards. However, the measurement of HO-1 activity is cumbersome and restricted to prolonged assays in cell lysates. A simple, easily used real-time activity probe would speed up research and may be an extremely useful diagnostic

A partnership between Joe Boyle in National Heart and Lung Institute and Nick Long in the Molecular Sciences Research Hub – particularly Ed Walter and Ying Ge - has designed, synthesised, and characterised entirely new fluorescent probe for HO-1. This molecule glows blue in the presence of ultraviolet light when Heme Oxygenase has acted on it. This was the first application of the Fluorescence Energy Transfer (FRET) breakapart approach to a small molecule metabolite (rather than an oligopeptide or oligonucleotide). In this, HO-1 removes a coumarin from precisely one position, preventing transfer of the fluorescence excitation from the coumarin to the porphyrin. Thereby, when the probe sees the enzyme, blue fluorescence emission results.

Analysis of HO-1 activity using Escherichia coli lysates and heme-stimulated human blood-derived macrophages overexpressing hHO-1 found that a 6-fold increase in emission intensity at 383 nm was observed following incubation with NADPH. The identities of the degradation products following catabolism were confirmed by MALDI-MS and LC−MS, showing that porphyrin catabolism was regioselective at the α-position. Finally, through the analysis of a control inactive analogues, we showed that close structural analogues of heme are required to maintain HO-1 activity. This work is springboarding design of new probes for HO-1 activity in the future, and we will develop and use in live applications in cells and in vivo models.

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