Quantification of Active Apohemoglobin Heme-Binding Sites via Dicyanohemin Incorporation

4 years ago

Quantification of Active Apohemoglobin Heme-Binding Sites via Dicyanohemin Incorporation

Ivan S. Pires, Donald A. Belcher, Andre F. Palmer

Apohemoglobin (apoHb) is produced by removing heme from hemoglobin (Hb). However, preparations of apoHb may contain damaged globins, which render total protein assays inaccurate for active apoHb quantification. Fortunately, apoHb heme-binding sites react with heme via the proximal histidine-F8 (His-F8) residue, which can be monitored spectrophotometrically. The bond between the His-F8 residue of apoHb and heme is vital for maintenance of fully functional and cooperative Hb. Additionally, most apoHb drug delivery applications facilitate hydrophobic drug incorporation inside the apoHb hydrophobic heme-binding pocket in which the His-F8 residue resides. This makes the His-F8 residue a proper target for apoHb activity quantification. In this work, dicyanohemin (DCNh), a stable monomeric porphyrin species, was used as a probe molecule to quantify active apoHb through monocyanohemin–His-F8 bond formation. ApoHb activity was quantified via the analysis of the 420 nm equilibrium absorbance of DCNh and apoHb mixtures. His-F8 saturation was determined by the presence of an inflection point from a plot of the 420 nm absorbance of a fixed concentration of apoHb against an increasing DCNh concentration. Various concentrations of a stock apoHb solution were tested to demonstrate the precision of the assay. The accuracy of the assay was assessed via spectral deconvolution, confirming His-F8 saturation at the inflection point. The effect of the heme-binding protein bovine serum albumin and precipitated apoHb on assay sensitivity was not significant. An analysis of the biophysical properties of reconstituted Hb confirmed heme-binding pocket activity. Taken together, this assay provides a simple and reliable method for determination of apoHb activity.

Publisher URL: http://dx.doi.org/10.1021/acs.biochem.7b00683

DOI: 10.1021/acs.biochem.7b00683