Related Experiment Video
Updated: Aug 18, 2026

Measurement of Heme Synthesis Levels in Mammalian Cells
Published on: July 9, 2015
Proton-linked subunit heterogeneity in ferrous nitrosylated human adult hemoglobin: an EPR study
Paolo Ascenzi1, Alessio Bocedi, Mauro Fasano
1Department of Biology, Interdepartmental Laboratory for Electron Microscopy, University Roma Tre, Viale Guglielmo Marconi 446, I-00146 Rome, Italy. ascenzi@uniroma3.it
Abstract:
The effect of pH on the X-band electron paramagnetic resonance (EPR) spectrum of ferrous nitrosylated human adult tetrameric hemoglobin (HbNO) as well as of ferrous nitrosylated monomeric alpha- and beta-chains has been investigated, at -163 degrees C. At pH 7.3, the X-band EPR spectrum of tetrameric HbNO and ferrous nitrosylated monomeric alpha- and beta-chains displays a rhombic shape. Lowering the pH from 7.3 to 3.0, tetrameric HbNO and ferrous nitrosylated monomeric alpha- and beta-chains undergo a transition towards a species characterized by a X-band EPR spectrum with a three-line splitting centered at 334mT. These pH-dependent spectroscopic changes may be taken as indicative of the cleavage, or the severe weakening, of the proximal HisF8-Fe bond. In tetrameric HbNO, the pH-dependent spectroscopic changes depend on the acid-base equilibrium of two apparent ionizing groups with pK(a) values of 5.8 and 3.8. By contrast, the pH-dependent spectroscopic changes occurring in ferrous nitrosylated monomeric alpha- and beta-chains depend on the acid-base equilibrium of one apparent ionizing group with pK(a) values of 4.8 and 4.7, respectively. The different pK(a) values for the proton-linked spectroscopic transition(s) of tetrameric HbNO and ferrous nitrosylated monomeric alpha- and beta-chains suggest that the quaternary assembly drastically affects the strength of the proximal HisF8-Fe bond in both subunits. This probably reflects a 'quaternary effect', i.e., structural changes in both subunits upon tetrameric assembly, which is associated to a relevant variation of functional properties (i.e., proton affinity).
Related Concept Videos
Gene Families
Occasionally these regions can be adapted to take on new roles within the organism, becoming novel genes...
Hemoglobin
When all four heme groups are bound to oxygen, the resulting molecule is called oxyhemoglobin. As a result, arterial blood...
¹H NMR Chemical Shift Equivalence: Homotopic and Heterotopic Protons
¹H NMR of Labile Protons: Deuterium (²H) Substitution
2D NMR: Overview of Heteronuclear Correlation Techniques
Electron Paramagnetic Resonance (EPR) Spectroscopy: Organic Radicals

![Protein Film Infrared Electrochemistry Demonstrated for Study of H2 Oxidation by a [NiFe] Hydrogenase](/_next/image?url=https%3A%2F%2Fcloudfront.jove.com%2FCDNSource%2Fteasers%2F55858.jpg&w=3840&q=50)