Constructing Genetic and Chemical Variants of Human Serum Albumin Reconstituted with Heme and Testing for Nitrite Reductase Activity

Open Access
Author:
Alfonso Castro, Alexandra
Area of Honors:
Chemistry
Degree:
Bachelor of Science
Document Type:
Thesis
Thesis Supervisors:
  • Mary Grace Ignacio Galinato, Thesis Supervisor
  • Michael A Campbell, Honors Advisor
Keywords:
  • Human serum albumin
  • HSA
  • Nitrite reductase
  • nitrite
  • nitric oxide
  • myoglobin
  • hemoglobin
  • rate constant
  • binding constant
  • UV-vis spectroscopy
  • reconstitution
  • heme
  • heme b
  • NiR
  • binding site
Abstract:
Human serum albumin (HSA) is abundantly present in our blood and is a repository to many molecules, such as heme. Previous studies have confirmed that HSA reconstituted with heme (HSA-heme) imitates the binding site of other heme enzymes such as myoglobin (Mb) and hemoglobin (Hb), except that an O(tyrosine) bonds to the Fe center of heme instead of a N(histidine) that is present in Mb and Hb. Although the primary function of Mb and Hb is to store and carry oxygen in the muscles and blood, respectively, they also have a secondary function of producing nitric oxide through the reduction of nitrite under hypoxic conditions. The aim of this project is to obtain imidazole complexes of HSA reconstituted with heme (HSA-hemeIm), and then test for their nitrite reductase (NiR) activity. This can be achieved by titrating the protein with imidazole compounds, specifically imidazole (Im), 1-methylimidazole (1-Im), and 2-methylimidazole (2-Im). Adding imidazole to HSA-heme may cause the formation of a N(imidazole) bond to Fe, providing a system that mimics the active site of Mb and Hb. HSA-heme is complexed with an imidazole derivative and reacted with nitrite. This reaction is monitored with UV-Vis spectroscopy. The spectrum is compared to that of HSA-heme imidazole derivative reacted with nitric oxide. These two spectra are very similar, indicating that HSA-hemeIm converts the nitrite into nitric oxide. Finally, the bimolecular rate constant obtained from the reaction between HSA-heme1-Im and nitrite is smaller than that obtained for the respective NiR reaction of wild-type (wt) HSA-heme or Mb.