Methods for the Extraction of Heme Prosthetic Groups from Hemoproteins

Kat Ellis-Guardiola; Jess Soule; Robert T Clubb

doi:10.21769/BioProtoc.4156

Methods for the Extraction of Heme Prosthetic Groups from Hemoproteins

Bio Protoc. 2021 Sep 20;11(18):e4156. doi: 10.21769/BioProtoc.4156.

Authors

Kat Ellis-Guardiola^{1

2}, Jess Soule¹, Robert T Clubb^{1

2

3}

Affiliations

¹ UCLA Department of Chemistry and Biochemistry, University of California, Los Angeles, 611 Charles Young Drive East, Los Angeles, CA 90095, USA.
² UCLA-DOE Institute of Genomics and Proteomics, University of California, Los Angeles, 611 Charles Young Drive East, Los Angeles, CA 90095, USA.
³ Molecular Biology Institute, University of California, Los Angeles, 611 Charles Young Drive East, Los Angeles, CA 90095, USA.

Abstract

Hemoproteins are widely researched because they contain redox-active heme prosthetic groups (iron + protoporphyrin IX) that enable them to perform a range of vital functions, acting as enzymes, participants in electron transfer reactions, or gas sensing, carrying, and storage proteins. While the heme prosthetic group is almost always essential for hemoprotein function, it is frequently desirable to remove it from the protein to enable biochemical or protein engineering studies. Obtaining high yields of the apo form of the hemoprotein can be challenging since high heme-protein binding affinities necessitate the use of harsh conditions to remove heme. In this Bio-Protocol, we present three chemical extraction methods that can be used to efficiently remove heme: methyl ethyl ketone extraction, acid-acetone precipitation, and on-column heme extraction. We also present protocols that can be used to quantitate the amount of residual heme bound to the protein after performing the extraction procedures.

Keywords: Acid-acetone precipitation; Heme extraction; Heme removal; Hemoprotein; Methyl ethyl ketone extraction; Porphyrin extraction; Porphyrin removal; heme.

Abstract

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