Measurement of O2 Binding by Sensory Hemeproteins

Marie A. Gilles-Gonzalez; Eduardo H.S. Sousa

doi:10.1007/978-1-0716-3080-8_2

Measurement of O₂ Binding by Sensory Hemeproteins

Marie A. Gilles-Gonzalez, Eduardo H.S. Sousa

Research output: Chapter in Book/Report/Conference proceeding › Chapter

Abstract

The discovery of an increasing number of proteins that function in the detoxification and sensing of gaseous ligands has renewed interest in hemeproteins. It is critical to measure the affinities of these proteins for ligands like O₂, CO, and NO, know with confidence when a protein is fully saturated with a specific ligand, and be able to estimate how well a ligand will compete against other ligands for a specific protein. Below we describe how to obtain an intact O₂-binding hemeprotein with a full complement of heme, how to evaluate the factors that can impact its affinity for O₂, and how to determine accurately the equilibrium and kinetic parameters K_d, k_on, and k_off for O₂ binding.

Original language	English (US)
Title of host publication	Methods in Molecular Biology
Publisher	Humana Press Inc.
Pages	11-25
Number of pages	15
DOIs	https://doi.org/10.1007/978-1-0716-3080-8_2
State	Published - 2023

Publication series

Name	Methods in Molecular Biology
Volume	2648
ISSN (Print)	1064-3745
ISSN (Electronic)	1940-6029

Keywords

Competition binding
Equilibrium binding
Heme proteins
O affinity
Stopped flow spectroscopy

ASJC Scopus subject areas

Molecular Biology
Genetics

Access to Document

10.1007/978-1-0716-3080-8_2

Cite this

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title = "Measurement of O2 Binding by Sensory Hemeproteins",

abstract = "The discovery of an increasing number of proteins that function in the detoxification and sensing of gaseous ligands has renewed interest in hemeproteins. It is critical to measure the affinities of these proteins for ligands like O2, CO, and NO, know with confidence when a protein is fully saturated with a specific ligand, and be able to estimate how well a ligand will compete against other ligands for a specific protein. Below we describe how to obtain an intact O2-binding hemeprotein with a full complement of heme, how to evaluate the factors that can impact its affinity for O2, and how to determine accurately the equilibrium and kinetic parameters Kd, kon, and koff for O2 binding.",

keywords = "Competition binding, Equilibrium binding, Heme proteins, O affinity, Stopped flow spectroscopy",

author = "Gilles-Gonzalez, {Marie A.} and Sousa, {Eduardo H.S.}",

note = "Funding Information: EHSS is thankful to CNPq (EHSS 308383/2018-4) and the National Institute of Science and Technology on Tuberculosis (INCT-TB, financed by CNPq/FAPERGS/CAPES/BNDES), and MAGG is grateful to the US National Science Foundation (grant no. MCB620531) for providing financial support. Publisher Copyright: {\textcopyright} 2023, The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.",

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AU - Sousa, Eduardo H.S.

N1 - Funding Information: EHSS is thankful to CNPq (EHSS 308383/2018-4) and the National Institute of Science and Technology on Tuberculosis (INCT-TB, financed by CNPq/FAPERGS/CAPES/BNDES), and MAGG is grateful to the US National Science Foundation (grant no. MCB620531) for providing financial support. Publisher Copyright: © 2023, The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.

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N2 - The discovery of an increasing number of proteins that function in the detoxification and sensing of gaseous ligands has renewed interest in hemeproteins. It is critical to measure the affinities of these proteins for ligands like O2, CO, and NO, know with confidence when a protein is fully saturated with a specific ligand, and be able to estimate how well a ligand will compete against other ligands for a specific protein. Below we describe how to obtain an intact O2-binding hemeprotein with a full complement of heme, how to evaluate the factors that can impact its affinity for O2, and how to determine accurately the equilibrium and kinetic parameters Kd, kon, and koff for O2 binding.

AB - The discovery of an increasing number of proteins that function in the detoxification and sensing of gaseous ligands has renewed interest in hemeproteins. It is critical to measure the affinities of these proteins for ligands like O2, CO, and NO, know with confidence when a protein is fully saturated with a specific ligand, and be able to estimate how well a ligand will compete against other ligands for a specific protein. Below we describe how to obtain an intact O2-binding hemeprotein with a full complement of heme, how to evaluate the factors that can impact its affinity for O2, and how to determine accurately the equilibrium and kinetic parameters Kd, kon, and koff for O2 binding.

KW - Competition binding

KW - Equilibrium binding

KW - Heme proteins

KW - O affinity

KW - Stopped flow spectroscopy

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