Quantification of Active Apohemoglobin Heme-Binding Sites via Dicyanohemin Incorporation

Abstract

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.

Novel manufacturing method for producing apohemoglobin and its biophysical properties

“…Various liganded forms of rHb were analyzed via UV‐Vis spectroscopy and compared against native Hb. The oxy‐rHb and oxyHb equilibrium binding curves were measured using a Hemox analyzer (TCS Scientific Corp., New Hope, PA) at 37°C as described in the literature (Eike & Palmer, ; Palmer et al, ; Pires et al, ; Rameez, Guzman et al, ). Spectral deconvolution software was developed in the Python programming language (Python Software Foundation Beaverton, OR) using the nonlinear least squares function curve_fit of the SciPy package to determine the fraction of various liganded forms of rHb that contribute to the final spectra of the rHb mixture.…”

“…The activity of the vacant heme-binding pocket of apoHb was determined via the dicyanohemin (DCNh) incorporation assay developed in our lab (Pires et al, 2017). The extinction coefficients of DCNh and rHbCN used were 85 and 114 mM -1 cm -1 , respectively (Eaton & Hofrichter, 1981;Hogness, Zscheile, Sidwell, & Barron, 1937).…”

Tangential flow filtration of haptoglobin

“…The concentration of apohemoglobin (apoHb) was estimated based on a modified version of the abridged dicyanohemin incorporation assay. 45 Briefly, Hp fractions were mixed with excess methemealbumin (heme bound to human serum albumin [HSA]) and the mixture left to react for 15 hours at room temperature. The change in absorbance of the reacted mixture compared to the initial fraction components was used to estimate the amount of heme exchanged from methemealbumin to apoHb.…”

Apohemoglobin-haptoglobin complexes attenuate the hypertensive response to low-molecular-weight polymerized hemoglobin

“…22 The heme-binding site activity of the resulting apoHb was quantified with a dicyanohemin assay. 21 The total amount of protein in solution was estimated based on the molar extinction coefficient of apoHb. 22…”

“…This molecule is able to scavenge free heme due to its specificity for heme and the highly hydrophobic nature of its vacant heme-binding pocket. 21,22 Combining apoHb with Hp results in a protein complex (apoHb-Hp) that may be able to scavenge both Hb and heme in the plasma. 18 Because of its ability to compartmentalize stroma-free Hb and scavenge free heme, we hypothesized that administration of the apoHb-Hp complex would reduce hypertension that results from the transfusion of LMW PolyHb.…”