Precise determination of heme binding affinity in proteins

Abstract

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 apoprotein 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.

Lipocalin Blc is a potential heme‐binding protein

“…Assuming 1 : 1 protein : ligand binding stoichiometry based on the obtained crystal structure, spectrophotometric titration yielded a K d of 1.4 ± 0.3 µ m . This value is comparable to those determined for full‐length wtBlc binding to fatty acids [14] or some other heme‐binding proteins like lipocalin α 1 ‐microglobulin [43], bacterial chlorite dismutase‐like protein HemQ [44], or some all‐helical hemoproteins like horseradish peroxidase [45]. The affinity for heme is lower than that of classical heme‐binding proteins like apohemoglobins and apomyoglobins ( K d on the order of 10 −14 m ) [46], some other β‐barrel proteins like nitrobindins ( K d on the order of 10 −12 m ) [47], or hemophores ( K d on the order of 10 −10 m ) [48].…”