Integrating UV-Vis Spectroscopy and Oxygen Optode for Accurate Determination of Oxygen Affinity of Proteins

Anoop Rama Damodaran; Ambika Bhagi-Damodaran

doi:10.1007/978-1-0716-3080-8_1

Integrating UV-Vis Spectroscopy and Oxygen Optode for Accurate Determination of Oxygen Affinity of Proteins

Anoop Rama Damodaran, Ambika Bhagi-Damodaran

Chemistry (Twin Cities)

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

2 Scopus citations

Abstract

Protein-based oxygen sensors exhibit a wide range of affinity values ranging from low nanomolar to high micromolar. How proteins utilize different metals, cofactors, and macromolecular structure to regulate their oxygen affinity (K_d) to a value that is appropriate for their biological function is an important question in biochemistry and microbiology. In this chapter, we describe a simple setup that integrates a UV-Vis spectrometer with an oxygen optode for direct determination of K_d of heme-containing oxygen sensors. We provide details on how to set up the assay, acquire and fit data for accurate K_d determination using Cs H-NOX (K_d = 23 ± 2 nM) as an example, and also discuss tips and tricks to make the assay work for other oxygen-binding proteins.

Original language	English (US)
Title of host publication	Methods in Molecular Biology
Publisher	Humana Press Inc.
Pages	1-10
Number of pages	10
DOIs	https://doi.org/10.1007/978-1-0716-3080-8_1
State	Published - 2023

Publication series

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

Bibliographical note

Publisher Copyright:
© 2023, The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.

Keywords

Gas sensing and signaling
Heme sensors
Ligand binding
Metalloproteins
Oxygen affinity
Spectroscopy
Bacterial Proteins/metabolism
Heme/chemistry
Molecular Structure
Oxygen/metabolism
Spectrum Analysis

PubMed: MeSH publication types

Journal Article
Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't

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10.1007/978-1-0716-3080-8_1

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title = "Integrating UV-Vis Spectroscopy and Oxygen Optode for Accurate Determination of Oxygen Affinity of Proteins",

abstract = "Protein-based oxygen sensors exhibit a wide range of affinity values ranging from low nanomolar to high micromolar. How proteins utilize different metals, cofactors, and macromolecular structure to regulate their oxygen affinity (Kd) to a value that is appropriate for their biological function is an important question in biochemistry and microbiology. In this chapter, we describe a simple setup that integrates a UV-Vis spectrometer with an oxygen optode for direct determination of Kd of heme-containing oxygen sensors. We provide details on how to set up the assay, acquire and fit data for accurate Kd determination using Cs H-NOX (Kd = 23 ± 2 nM) as an example, and also discuss tips and tricks to make the assay work for other oxygen-binding proteins.",

keywords = "Gas sensing and signaling, Heme sensors, Ligand binding, Metalloproteins, Oxygen affinity, Spectroscopy, Bacterial Proteins/metabolism, Heme/chemistry, Molecular Structure, Oxygen/metabolism, Spectrum Analysis",

author = "Damodaran, {Anoop Rama} and Ambika Bhagi-Damodaran",

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language = "English (US)",

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AU - Damodaran, Anoop Rama

AU - Bhagi-Damodaran, Ambika

PY - 2023

Y1 - 2023

N2 - Protein-based oxygen sensors exhibit a wide range of affinity values ranging from low nanomolar to high micromolar. How proteins utilize different metals, cofactors, and macromolecular structure to regulate their oxygen affinity (Kd) to a value that is appropriate for their biological function is an important question in biochemistry and microbiology. In this chapter, we describe a simple setup that integrates a UV-Vis spectrometer with an oxygen optode for direct determination of Kd of heme-containing oxygen sensors. We provide details on how to set up the assay, acquire and fit data for accurate Kd determination using Cs H-NOX (Kd = 23 ± 2 nM) as an example, and also discuss tips and tricks to make the assay work for other oxygen-binding proteins.

AB - Protein-based oxygen sensors exhibit a wide range of affinity values ranging from low nanomolar to high micromolar. How proteins utilize different metals, cofactors, and macromolecular structure to regulate their oxygen affinity (Kd) to a value that is appropriate for their biological function is an important question in biochemistry and microbiology. In this chapter, we describe a simple setup that integrates a UV-Vis spectrometer with an oxygen optode for direct determination of Kd of heme-containing oxygen sensors. We provide details on how to set up the assay, acquire and fit data for accurate Kd determination using Cs H-NOX (Kd = 23 ± 2 nM) as an example, and also discuss tips and tricks to make the assay work for other oxygen-binding proteins.

KW - Gas sensing and signaling

KW - Heme sensors

KW - Ligand binding

KW - Metalloproteins

KW - Oxygen affinity

KW - Spectroscopy

KW - Bacterial Proteins/metabolism

KW - Heme/chemistry

KW - Molecular Structure

KW - Oxygen/metabolism

KW - Spectrum Analysis

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T3 - Methods in Molecular Biology

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BT - Methods in Molecular Biology

PB - Humana Press Inc.

ER -

Integrating UV-Vis Spectroscopy and Oxygen Optode for Accurate Determination of Oxygen Affinity of Proteins

Abstract

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