title: Angiotensin converting enzyme immobilized on magnetic beads as a tool for ligand fishing.
abstract: Angiotensin converting enzyme (ACE) presents an important role in blood pressure regulation, since that converts angiotensin I to the vasoconstrictor angiotensin II. Some commercially available ACE inhibitors are captopril, lisinopril and enalapril; due to their side effects, naturally occurring inhibitors have been prospected. In order to endorse this research field we have developed a new tool for ACE ligand screening. To this end, ACE was extracted from bovine lung, purified and chemically immobilized in modified ferrite magnetic beads (ACE-MBs). The ACE-MBs have shown a Michaelian kinetic behavior towards hippuryl-histidyl-leucine. Moreover, as proof of concept, the ACE-MBs was inhibited by lisinopril with a half maximal inhibitory concentration (IC50) of 10nM. At the fishing assay, ACE-MBs were able not only to fish out the reference inhibitor, but also one peptide from a pool of tryptic digested BSA. In conclusion, ACE-MBs emerge as new straightforward tool for ACE kinetics determination, inhibition and binder screening.
authors.affiliation: SEPARARE Núcleo de Pesquisa em Cromatografia, Departamento de Química, Universidade Federal de São Carlos, Cx. Postal 676, São Carlos, 13565-905, SP, Brazil.
authors.forename: Fernando G
authors.initials: FG
authors.lastname: de Almeida
authors.affiliation: SEPARARE Núcleo de Pesquisa em Cromatografia, Departamento de Química, Universidade Federal de São Carlos, Cx. Postal 676, São Carlos, 13565-905, SP, Brazil.
authors.forename: Kenia L
authors.initials: KL
authors.lastname: Vanzolini
authors.affiliation: SEPARARE Núcleo de Pesquisa em Cromatografia, Departamento de Química, Universidade Federal de São Carlos, Cx. Postal 676, São Carlos, 13565-905, SP, Brazil. Electronic address: quezia@pq.cnpq.br.
authors.forename: Quezia B
authors.initials: QB
authors.lastname: Cass
mesh_terms: D000806:Angiotensin-Converting Enzyme Inhibitors / Q000737:chemistry* / Q000494:pharmacology; D000809:Angiotensins / Q000737:chemistry; D000818:Animals; D002216:Captopril / Q000737:chemistry; D002417:Cattle; D002853:Chromatography, Liquid; D004656:Enalapril / Q000737:chemistry; D004800:Enzymes, Immobilized / Q000737:chemistry*; D020128:Inhibitory Concentration 50; D007501:Iron / Q000737:chemistry; D007700:Kinetics; D008024:Ligands; D017706:Lisinopril / Q000737:chemistry; D008168:Lung / Q000378:metabolism; D058185:Magnetite Nanoparticles / Q000737:chemistry; D007703:Peptidyl-Dipeptidase A / Q000737:chemistry*; D015203:Reproducibility of Results; D013499:Surface Properties; D053719:Tandem Mass Spectrometry; D014357:Trypsin / Q000737:chemistry
chemical_list: D000806:Angiotensin-Converting Enzyme Inhibitors; D000809:Angiotensins; D004800:Enzymes, Immobilized; D008024:Ligands; D058185:Magnetite Nanoparticles; D004656:Enalapril; D002216:Captopril; D007501:Iron; D017706:Lisinopril; D007703:Peptidyl-Dipeptidase A; D014357:Trypsin
keywords: ACE activity assay; ACE immobilization; ACE purification; LC–MS/MS; Magnetic nanoparticles; Peptide binding assay