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The assessment of interaction between eNOS and Alpha hemoglobin by Bio‐layer Interferometry
Author(s) -
Ma Dongying,
Ackerman Hans
Publication year - 2018
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2018.32.1_supplement.652.14
Subject(s) - enos , globin , hemoglobin , chemistry , dissociation constant , protein subunit , alpha (finance) , affinity chromatography , microbiology and biotechnology , biochemistry , nitric oxide synthase , receptor , biology , enzyme , gene , medicine , construct validity , nursing , patient satisfaction
Alpha globin, a subunit of red blood cell hemoglobin, is also expressed in human and mouse resistance artery endothelial cells. Qualitative protein‐protein interaction methods have been used to identify a macromolecular complex that forms between alpha globin and endothelial nitric oxide synthase (eNOS). The aim of this study is to characterize the interaction between alpha globin and eNOS quantitatively using bio‐layer interferometry (BLI). To this end, alpha globin was purified from whole hemoglobin using ion exchange chromatography and the purity was assessed by mass spectrometry. The oxygenase of eNOS (eNOSox) was expressed in PGEX vector and UT5600 E. coli system. Further, the ForteBio Octet system was used to identify the binding characterization of purified alpha globin with eNOS, with its molecular chaperon a‐Hemoglobin stabilizing protein (AHSP) as control. The results showed that the affinity constant KD between alpha globin and AHSP is 1.09×10 −7 M, which was calculated using the association rate ka (2.28×10 4 M −1 s −1 ) and dissociation rate (2.49×10 −3 s −1 ). The affinity constant KD between alpha globin and eNOSox is 1.31×10 −6 M, which was calculated using the association rate ka (1.16×10 3 M −1 s −1 ) and dissociation rate (1.51×10 −3 s −1 ). Smaller affinity constant indicates greater affinity of analyte to ligand. Thus, the interaction between Alpha globin and AHSP is tighter than the interaction between alpha globin and eNOSox. However, the lower kd value for alpha‐globin and eNOSox indicates the slower dissociation, compared to dissociation from AHSP. This result will help us to better understand the molecular mechanisms of the regulation of alpha globin on eNOS function, and provide foundation for the development of chemical drug compounds which target specifically on this interaction. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .