Oxygen binding to heme proteins in solution, encapsulated in silica gels, and in the crystalline state

Methods in Enzymology
Luca RondaAndrea Mozzarelli

Abstract

The determination of accurate oxygen-binding curves for heme-containing proteins is a demanding task. In fact, great care is required in the (i) preparation of accurate gas mixtures at defined oxygen partial pressures, (ii) precise measurement of changes in protein absorbance, (iii) calculation of the fraction of oxygen-containing sites, and (iv) analysis of the dependence of fractional saturation on oxygen pressure using phenomenological or model-dependent equations. Over the years, methods have been developed for the determination of oxygen-binding curves based either on discrete steps in oxygen partial pressure ("static" method) or on continuous variations ("dynamic" method). This work presents a novel, versatile setup that allows one to determine oxygen-binding curves for heme and nonheme proteins in solution, encapsulated in wet, nanoporous silica gels, in the crystalline state, and for hemoglobin within single red blood cells. The apparatus is composed of a tandem of high-precision gas mixture generators and either an equilibration chamber coupled to a spectrophotometer cuvette or a gas-tight flow cell, placed on the stage of a microspectrophotometer, for immobilized samples down to a few micrometers in size.

Citations

Sep 13, 2012·Sensors·Cindy AstJoost T van Dongen
Sep 3, 2013·The FEBS Journal·Marialaura MarchettiAndrea Mozzarelli
Jun 17, 2020·Biomaterials Science·Chris E CooperBrandon J Reeder
Sep 12, 2020·Computational and Structural Biotechnology Journal·Daniela GiordanoCinzia Verde
Jun 19, 2009·Bioconjugate Chemistry·Dario CacciaAndrea Mozzarelli
Dec 28, 2010·Biochimica Et Biophysica Acta·Luca RondaAndrea Mozzarelli

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