Abstract
Ferrochelatase assay or heme content was frequently determined by the pyridine-hemochrome method, but this was disadvantageous because of low coefficient of pyridine-hemochrome and toxicity of pyridine. In plants, heme is biosynthesized in chloroplast and distributed to various organelles. To elucidate heme trafficking, a safe and sensitive heme assay is necessary. Here, we developed an sensitive assay using reconstitution with apo-horseradish peroxidase (HRP), and determination of peroxidase activity by measuring chemiluminescence. Active peroxidase was reconstituted within 30 min, and stable chemiluminescence was obtained 30 min after reaction. This method was linear with heme concentration and was extremely sensitive with detection limit as little as 10 pM. Various porphyrins and metalloporphyrins did not affect the chemiluminescence. The luminol oxidizing effect of Fe2+ was less than 3 magnitude of heme. Application of this method, such as heme efflux from tetrapyrrole-binding protein, the ferrochelatase assay, and heme content in organelles are being analyzed.
