title: Direct assay of enzymes in heme biosynthesis for the detection of porphyrias by tandem mass spectrometry. Uroporphyrinogen decarboxylase and coproporphyrinogen III oxidase.
abstract: We report new assays of enzymes uroporphyrinogen decarboxylase (UROD) and coproporphyrinogen III oxidase (CPO) in the heme biosynthetic pathway. The assays were developed for use in clinical diagnostics of inherited disorders porphyria cutanea tarda and hereditary coproporphyria, respectively. Electrospray ionization tandem mass spectrometry is used to monitor the decarboxylation of pentaporphyrinogen I or uroporphyrinogen III catalyzed by UROD and to determine the enzyme activity in human erythrocytes by measuring the production of coproporphyrinogen I or III. The Km value for pentaporphyrinogen I was measured as 0.17 +/- 0.03 microM. A mass spectrometric assay was also developed for the two-step decarboxylative oxidation of coproporphyrinogen III to protoporphyrinogen IX catalyzed by CPO in mitochondria from human lymphocytes (Km = 0.066 +/- 0.009 microM). The assays show good reproducibility, use simple workup by liquid-liquid extraction of enzymatic products, and employ commercially available substrates and internal standards.
authors.affiliation: Departments of Chemistry, Pediatrics, and Biochemistry, University of Washington, Seattle, Washington 98195-1700, USA.
authors.forename: Yuesong
authors.initials: Y
authors.lastname: Wang
authors.forename: Paula
authors.initials: P
authors.lastname: Gatti
authors.forename: Martin
authors.initials: M
authors.lastname: Sadílek
authors.forename: C Ronald
authors.initials: CR
authors.lastname: Scott
authors.forename: Frantisek
authors.initials: F
authors.lastname: Turecek
authors.forename: Michael H
authors.initials: MH
authors.lastname: Gelb
mesh_terms: D000332:Aerobiosis; D003304:Coproporphyrinogen Oxidase / Q000032:analysis* / Q000378:metabolism; D006418:Heme / Q000032:analysis* / Q000096:biosynthesis / Q000737:chemistry*; D006801:Humans; D015394:Molecular Structure; D011164:Porphyrias / Q000175:diagnosis* / Q000378:metabolism*; D013379:Substrate Specificity; D053719:Tandem Mass Spectrometry / Q000379:methods*; D014575:Uroporphyrinogen Decarboxylase / Q000032:analysis* / Q000378:metabolism
chemical_list: D006418:Heme; D003304:Coproporphyrinogen Oxidase; D014575:Uroporphyrinogen Decarboxylase