Abstract
Heme is an iron-containing porphyrin which acts as a prosthetic group in several enzymes involved in disparate functions, such as respiration and H2O2-scavenging. Escherichia coli is able to produce heme endogenously since it contains all the enzymes involved in the nine-step biosynthesis pathway, which in absence of stress and in iron-replete media proceeds unabated. However, we recently showed that two steps are affected by H2O2 stress (Mancini and Imlay, 2015). To compensate, two enzymes, namely the ferrochelatase (HemH) and an isozyme of coproporphyrinogen III oxidase (HemF), are activated by the H2O2-responsive regulator OxyR. Genetic mutations that block either adaptation cause the intracellular accumulation of protoporphyrin IX and coproporphyrinogen III, the substrates of HemH and HemF, respectively. We here describe a method used to extract and quantify protoporphyrin IX and coproporphyrin III, the product of the spontaneous oxidation of coproporphyrinogen III.
Keywords: Heme, Protoporphyrin, Protoporphyrinogen, Ferrochelatase
Materials and Reagents
Equipment
For porphyrin extraction
For the LC/MS/MS analysis
Procedure
For the porphyrin extraction (adapted from Nakayashiki and Inokuchi, 1997)
Representative data
Figure 1. MRM chromatograms of protoporphyrin IX. Intracellular accumulation of protoporphyrin IX in H2O2-stressed cells (Hpx2-) is exacerbated by the lack of hemH activation [Hpx2- hemH(NI)]. 1 µg protoporphyrin IX was included as reference standard. Figure 2. MRM chromatograms of coproporphyrin III. Intracellular coproporphyrinogen III accumulates in H2O2-stressed cells lacking hemF (Hpx2- ∆hemF), with levels comparable to those detected in cells lacking both the coproporphyrinogen III oxidase isozymes (∆hemN ∆hemF). 1 µg coproporphyrin III was included as reference standard.
Notes
Upon exposure to air all porphyrinogens autoxidize to porphyrins. Therefore, coproporphyrin III and not coproporphyrinogen III (the actual substrate of the two coproporphyrinogen III oxidase isozymes HemN and HemF) can be analyzed and quantified by LC/MS/MS. Similarly, protoporphyrinogen IX, the substrate of the penultimate enzyme of the heme biosynthesis pathway, namely protoporphyrinogen IX oxidase, spontaneously oxidizes to protoporphyrin IX. Therefore, protoporphyrin IX levels should reflect the sum of protoporphyrinogen IX and protoporphyrin IX.
Acknowledgments
This work was supported by grant GM49640 from the National Institutes of Health and by award number PBBEP3_139397 from the Swiss National Science Foundation. The protocol for the porphyrin extraction was adapted from Nakayashiki and Inokuchi (1997).
References
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