ApoHRP-based assay to measure intracellular regulatory heme

Abstract

The majority of the heme-binding proteins possess a “heme-pocket” that stably binds with 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 independently from the total heme (TH). The current study describes and validates a new method to measure intracellular RH. The 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 from 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.

Regulation of intracellular heme trafficking revealed by subcellular reporters

“…Given that the affinity of heme to intracellular and extracellular heme-binding proteins varies from 1 μM to <1 pM (3,73), and the K m of heme oxygenase-1 is ∼1 μM, cytosolic labile heme concentrations are likely to be less than 1 μM (33). Because heme can be transferred from one protein to another with a higher heme affinity (74,75), in vitro reconstitution of purified apoHRP, which has a heme affinity lower than housekeeping proteins that bind protoheme or heme b, has been previously used to determine labile heme in IMR90 lung fibroblast cell extracts (34,35). This method estimated labile heme to be 614 ± 214 nM, in close approximation to our estimate of 433 ± 125 nM in HEK293 cells.…”

“…This in vitro HRP reconstitution method was found to be more sensitive than the traditional pyridine hemochromogen method (39), with minimal interference from fluorescent porphyrins (40). However, a major drawback for this method was that the biological material must first be disrupted and then mixed with apoHRP to measure the conversion of apo to holo, thereby preventing analysis of subcellular heme distribution (34,35). HRP variants were created by using specific subcellular targeting sequences to direct the expressed protein to the cytosol, mitochondrial matrix, peroxisome, endoplasmic reticulum (ER), Golgi complex, and plasma membrane (PM; GPI anchored) (SI Appendix, Table S1).…”

“…This value is weaker than constituent heme proteins and enzymes (e.g., globins, cytochromes, and catalase) that have heme affinity ranges between 10 −12 and 10 −15 M (33), making HRP an ideal reporter for labile heme. Studies have used purified apoHRP to bind bioavailable heme in cell-free extracts as a way to measure regulatory heme (34)(35)(36)(37)(38). This in vitro HRP reconstitution method was found to be more sensitive than the traditional pyridine hemochromogen method (39), with minimal interference from fluorescent porphyrins (40).…”

Heme Mobilization in Animals: A Metallolipid’s Journey

“…9 Another key development is the generation of fluorescent and enzymatic reporters to probe the dynamics and distribution of heme in cells and animals. 11–13 …”

Rapid and Sensitive Quantitation of Heme in Hemoglobinized Cells

“…With the exception of the cyanmethemoglobin assay, these approaches infer hemoglobin concentrations from measurements of heme. More recently, chemiluminescent-based methods based on horseradish peroxidase biochemistry have also been developed to quantitate total cellular heme (10) and regulatory heme (11). We have previously relied on the pyridine hemochromogen and fluorescence assays to determine cellular heme and hemoglobin concentrations as described elsewhere (12,13).…”