Chemiluminescent-based method for heme determination by reconstitution with horseradish peroxidase apo-enzyme

Abstract

Heme, an essential molecule with various biological functions, is widely distributed in all eukaryotic cells. In addition to the electron transport cytochrome of chloroplasts and mitochondria, the cytosol contains relatively abundant hemoproteins, including catalase, cytochrome P450, and peroxidase. The mechanism by which the cytosolic proteins receive their hemes from the site of heme synthesis is not well understood in either plant or animal cells. To determine the eZux of heme from such organelles, an extremely sensitive heme assay is needed. The currently available methods for quantiWcation of heme are disadvantageous because of the low coeYcient of pyridine–hemochrome and the toxicity of pyridine [1]. Although several sensitive colorimetric methods have been developed [2–5], the detection limits of heme were more than nanomolar, even in the most sensitive assays [3–5]. In this study, we developed an extremely sensitive assay for heme based on the ability of horseradish peroxidase (HRP) 1 apo-enzyme to reconstitute with heme to form an active enzyme. The assay is a chemiluminescent-based method and requires only one step of reagent addition.