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Precise determination of heme binding affinity in proteins.

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journal contribution
posted on 20.05.2019, 10:10 by Galvin C.-H. Leung, Simon S.-P. Fung, Nicholas R. B. Dovey, Emma L. Raven, Andrew J. Hudson
Accumulating evidence suggests a new role for cellular heme as a signalling molecule, in which interactions with target proteins are more transient than found with traditionally-defined hemoproteins. To study this role, a precise method is needed for determining the heme-binding affinity (or dissociation constant, Kd). Estimates of Kd are commonly made following a spectrophotometric titration of an apo-protein with hemin. An impediment to precise determination is, however, the challenge in discriminating between the Soret absorbance for the product (holo-protein) and that for the titrant (hemin). An altogether different approach has been used in this paper to separate contributions made by these components to absorbance values. The pure component spectra and concentration profiles are estimated by a multivariate curve-resolution (MCR) algorithm. This approach has significant advantages over existing methods. First, a more precise determination of Kd can be made as concentration profiles for all three components (apo-protein/holo-protein/hemin) are determined and can be simultaneously fitted to a theoretical-binding model. Second, an absorption spectrum for the holo-protein is calculated. This is a unique advantage of MCR and attractive for investigating proteins in which the nature of heme binding has not, hitherto, been characterised because the holo-protein spectrum provides information on the interaction.

Funding

The research was supported by the Leverhulme Trust (RPG-2016-397) awarded to AJH and ELR.

History

Citation

Analytical Biochemistry, 2019, 572, pp. 45-51

Author affiliation

/Organisation/COLLEGE OF SCIENCE AND ENGINEERING/Department of Chemistry

Version

AM (Accepted Manuscript)

Published in

Analytical Biochemistry

Publisher

Elsevier Masson

eissn

1096-0309

Acceptance date

22/02/2019

Copyright date

2019

Publisher version

https://www.sciencedirect.com/science/article/pii/S0003269719300168?via=ihub

Notes

The file associated with this record is under embargo until 12 months after publication, in accordance with the publisher's self-archiving policy. The full text may be available through the publisher links provided above.;Supplementary data to this article can be found online at https://doi.org/10.1016/j.ab.2019.02.021

Language

en

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