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.