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An enzymatic method for precise oxygen affinity measurements over nanomolar-to-millimolar concentration regime

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Abstract

Oxygen affinity is an important property of metalloproteins that helps elucidate their reactivity profile and mechanism. Heretofore, oxygen affinity values were determined either using flash photolysis and polarography techniques that require expensive instrumentation, or using oxygen titration methods which are erroneous at low nanomolar and at high millimolar oxygen concentrations. Here, we describe an inexpensive, easy-to-setup, and a one-pot method for oxygen affinity measurements that uses the enzyme chlorite dismutase (Cld) as a precise in situ oxygen source. Using this method, we measure thermodynamic and kinetic oxygen affinities (Kd and KM) of different classes of heme and non-heme metalloproteins involved in oxygen transport, sensing, and catalysis. The method enables oxygen affinity measurements over a wide concentration range from 10 nM to 5 mM which is unattainable by simply diluting oxygen-saturated buffers. In turn, we were able to precisely measure oxygen affinities of a model set of eight different metalloproteins with affinities ranging from 48 ± 3 nM to 1.18 ± 0.03 mM. Overall, the Cld method is easy and inexpensive to set up, requires significantly lower quantities of protein, enables precise oxygen affinity measurements, and is applicable for proteins exhibiting nanomolar-to-millimolar affinity values.

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Acknowledgements

This work was supported by start-up funds from the Regents of University of Minnesota. The authors are grateful to Prof. J. D. Lipscomb, Dr. Rahul Banerjee and Dr. Melanie Rogers for providing enzymes HPCD, hemerythrin and lab space/equipment for few experiments described in this work. We are grateful to Prof. Carsten Krebs and Prof. J. Martin Bollinger Jr. for the Cld plasmid and Prof. Yi Lu for sw WTMb plasmid. We thank Prof. Michael A. Marletta and Dr. Christopher Lemon for gifting the H-NOX enzyme and valuable discussions. We thank Addgene for providing GroESL plasmid. We also thank Profs. J. D. Lipscomb, Connie Lu and Yi Lu for helpful comments to improve the quality of this manuscript.

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  1. Department of Chemistry, University of Minnesota, Twin Cities, Minneapolis, MN, 55455, USA

    Ria Sanyal & Ambika Bhagi-Damodaran

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  1. Ria Sanyal
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Correspondence to Ambika Bhagi-Damodaran.

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Sanyal, R., Bhagi-Damodaran, A. An enzymatic method for precise oxygen affinity measurements over nanomolar-to-millimolar concentration regime. J Biol Inorg Chem 25, 181–186 (2020). https://doi.org/10.1007/s00775-019-01750-6

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