Quantification of Apohemoglobin
Other Titles:
Quantification of Apohemoglobin via Dicyanohemin IncorporationQuantification of Active Apohemoglobin Heme-Binding Sites via Dicyanohemin Incorporation
Publisher:
The Ohio State UniversitySeries/Report no.:
The Ohio State University. William G. Lowrie Department of Chemical and Biomolecular Engineering Honors Theses; 2019Abstract:
Apohemoglobin (apoHb) is produced by removing heme from hemoglobin (Hb). Unfortunately, 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 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 was not significant on assay sensitivity. Functional reconstituted Hb (rHb) was made from analysis of the biophysical properties of rHb confirmed heme-binding pocket activity. Taken together, this assay provides a simple and reliable method for determination of apoHb activity. This activity will aid in future apoHb studies which may focus on apoHb encapsulation of photosensitive particles for photodynamic therapy, drugs for drug transport and/or targeted drug delivery.
Academic Major:
Academic Major: Chemical Engineering
Sponsors:
Ohio State University Office of Undergraduate Research & Creative Inquiry Summer Research Fellowship
College of Engineering Honors Research Fellowship
National Institutes of Health Grants R56-HL123015, R01-HL126945 and R01-EB021926
College of Engineering Honors Research Fellowship
National Institutes of Health Grants R56-HL123015, R01-HL126945 and R01-EB021926
Embargo:
No embargo
Type:
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