Precise determination of heme binding affinity in proteins

Galvin C.H. Leung, Simon S.P. Fung, Nicholas R.B. Dovey, Emma L. Raven*, Andrew J. Hudson

*Corresponding author for this work

Research output: Contribution to journalArticle (Academic Journal)peer-review

6 Citations (Scopus)
171 Downloads (Pure)


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, K d ). Estimates of K d 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 K d 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.

Original languageEnglish
Pages (from-to)45-51
Number of pages7
JournalAnalytical Biochemistry
Early online date23 Feb 2019
Publication statusPublished - 1 May 2019


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