title: High throughput heme assay by detection of chemiluminescence of reconstituted horseradish peroxidase.
abstract: In living organisms, heme is an essential molecule for various biological functions. Recent studies also suggest that heme functions as organelle-derived signal that regulates fundamental cell processes. Furthermore, estimation of heme is widely used for studying various blood disorders. In this regard, development of a rapid, sensitive, and high throughput heme assay has been sought. The most frequently used method of measuring heme by pyridine hemochrome is time, labor, and material intensive, and therefore limiting in its utility for large scale, high throughput analysis. Recently, we reported alternative method that is sensitive and specific to heme, which is based on the ability of horseradish peroxidase (HRP) apo-enzyme to reconstitute with heme to form an active holo-enzyme. Here, we developed high throughput heme assay by performing reactions on multi-well plate with highly sensitive chemiluminescence detection reagents. Detection of chemiluminescence in charged coupled device (CCD)-based gel doc apparatus enables simultaneous measurement of multiple samples. Furthermore, the high sensitivity of this assay allowed a direct measurement of heme in solvent extracts after dilution. This assay is sensitive, quick, provides a large dynamic range, and is well suited for large-scale analysis of heme extracted from minute amount of samples.
authors.affiliation: Graduate School of Arts and Sciences, University of Tokyo, Meguro-ku, Tokyo 153-8902, Japan.
authors.forename: Shigekazu
authors.initials: S
authors.lastname: Takahashi
authors.forename: Tatsuru
authors.initials: T
authors.lastname: Masuda
mesh_terms: D001051:Apoenzymes / Q000378:metabolism; D031215:Armoracia / Q000201:enzymology*; D006418:Heme / Q000032:analysis* / Q000378:metabolism*; D006735:Horseradish Peroxidase / Q000032:analysis / Q000378:metabolism*; D008163:Luminescent Measurements / Q000191:economics / Q000379:methods*; D012680:Sensitivity and Specificity; D013997:Time Factors
chemical_list: D001051:Apoenzymes; D006418:Heme; D006735:Horseradish Peroxidase