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ApoHRP-based assay to measure intracellular regulatory heme

Content Provider	Royal Society of Chemistry (RSC)
Author	Atamna, Hani Brahmbhatt, Marmik Dhahbi, Joseph M. Atamna, Wafa Shanower, Gregory A.
Copyright Year	2015
Abstract	The majority of the heme-binding proteins possess a “heme-pocket” that stably binds to heme. Usually known as housekeeping heme-proteins, they participate in a variety of metabolic reactions (e.g., catalase). Heme also binds with lower affinity to the “Heme-Regulatory Motifs” (HRM) in specific regulatory proteins. This type of heme binding is known as exchangeable or regulatory heme (RH). Heme binding to HRM proteins regulates their function (e.g., Bach1). Although there are well-established methods for assaying total cellular heme (e.g., heme-proteins plus RH), currently there is no method available for measuring RH independent of the total heme (TH). The current study describes and validates a new method to measure intracellular RH. This method is based on the reconstitution of apo-horseradish peroxidase (apoHRP) with heme to form holoHRP. The resulting holoHRP activity is then measured with a colorimetric substrate. The results show that apoHRP specifically binds RH but not with heme from housekeeping heme-proteins. The RH assay detects intracellular RH. Furthermore, using conditions that create positive (hemin) or negative (N-methyl protoporphyrin IX) controls for heme in normal human fibroblasts (IMR90), the RH assay shows that RH is dynamic and independent of TH. We also demonstrated that short-term exposure to subcytotoxic concentrations of lead (Pb), mercury (Hg), or amyloid-β (Aβ) significantly alters intracellular RH with little effect on TH. In conclusion the RH assay is an effective assay to investigate intracellular RH concentration and demonstrates that RH represents ∼6% of total heme in IMR90 cells.
Starting Page	309
Ending Page	321
Page Count	13
File Format	HTM / HTML PDF
ISSN	17565901
Volume Number	7
Issue Number	2
Journal	Metallomics
DOI	10.1039/c4mt00246f
Language	English
Publisher	Royal Society of Chemistry
Access Restriction	Open
Subject Keyword	HRM Hemin Amyloid beta Catalase Heme Methylmercury Lead Peroxidase Protoporphyrin IX BACH1
Content Type	Text
Resource Type	Article
Subject	Chemistry Medicine Metals and Alloys Biochemistry Biomaterials Biophysics

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