title: Nitroavidin as a ligand for the surface capture and release of biotinylated proteins.
abstract: Discussed here is the preparation, detailed purification, and evaluation of nitroavidin as a ligand for surface capture and release of biotinylated proteins. Avidin from chicken egg white was nitrated using dilute tetranitromethane solutions. UV-vis spectroscopy was used to show decreased binding of the biotin analogue, 2-(4'-hydroxyazobenzene)benzoic acid, HABA, to nitroavidin compared to binding of HABA to native avidin. From enzyme-linked immunosorbent assay (ELISA)-based assays of the modified avidin, it was found that there are approximately three tyrosine residues converted to nitrotyrosine out of the total four tyrosine residues in the protein tetramer. For the first time, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) was used to demonstrate the point of nitration in nitroavidin as that of the tyrosine associated with the binding of biotin (Y33). From surface plasmon resonance spectroscopy (SPR) experiments, it is shown that biotin has less binding propensity to immobilized nitroavidin (K(D) = 4.4 +/- 1.9 x 10(-6) M) than immobilized avidin (K(D) < or = 10(-11) M). Importantly, the use of pH 10 carbonate buffer as eluent resulted in facile release of bound biotin from the nitroavidin-functionalized surfaces, allowing for readily regenerated biotin capture surfaces (reversible binding surfaces). These outcomes are important for the development of protein concentration methods directed at isolation of select proteins from a large population using gentle target protein isolation/release conditions.
authors: Department of Chemistry and Center for Biomodular Multi-scale Systems, Louisiana State University, 232 Choppin Hall, Baton Rouge, Louisiana 70803-1804, USA.
authors: Jowell G
authors: JG
authors: Bolivar
authors: Steven A
authors: SA
authors: Soper
authors: Robin L
authors: RL
authors: McCarley
mesh_terms: D000818:Animals; D001360:Avidin / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism*; D001391:Azo Compounds / Q000378:metabolism; D001710:Biotin / Q000031:analogs & derivatives; D019921:Biotinylation; D002645:Chickens; D055767:Immobilized Proteins / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism; D008024:Ligands; D009574:Nitro Compounds / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism*; D010063:Ovum / Q000737:chemistry; D010455:Peptides / Q000032:analysis / Q000737:chemistry; D011485:Protein Binding; D011506:Proteins / Q000737:chemistry / Q000378:metabolism*; D020349:Surface Plasmon Resonance; D013774:Tetranitromethane; D014443:Tyrosine / Q000031:analogs & derivatives / Q000032:analysis / Q000737:chemistry
chemical_list: D001391:Azo Compounds; D055767:Immobilized Proteins; D008024:Ligands; D009574:Nitro Compounds; D010455:Peptides; D011506:Proteins; D001360:Avidin; C002437:2-(4'-hydroxyazobenzene)benzoic acid; C002744:3-nitrotyrosine; D014443:Tyrosine; D001710:Biotin; D013774:Tetranitromethane