title: New approach for natural products screening by real-time monitoring of hemoglobin hydrolysis using quartz crystal microbalance.
abstract: The hemoglobin (Hb) released from erythrocytes is a primary nutritive component for many blood-feeding parasites. The aspartic protease cathepsin D is a hemoglobinase that is involved in the Hb degradation process and is considered an interesting target for chemotherapy intervention. However, traditional enzymatic assays for studying Hb degradation utilize spectrophotometric techniques, which do not allow real-time monitoring and can present serious interference problems. Herein, we describe a biosensor using simple approach for the real-time monitoring of Hb hydrolysis as well as an efficient screening method for natural products as enzymatic inhibitors using a quartz crystal microbalance (QCM) technique. Hemoglobin was anchored on the quartz crystal surface using mixed self-assembled monolayers. The addition of the enzyme caused a mass change (frequency shift) due to Hb hydrolysis, which was monitored in real time. From the frequency change patterns of the Hb-functionalized QCM, we evaluated the enzymatic reaction by determining the kinetic parameters of product formation (k(cat)). The QCM enzymatic assay using immobilized human Hb was shown to be an excellent approach for screening possible inhibitors in complex mixtures, opening up a new avenue for the discovery of novel inhibitors.
authors: Department of Chemistry, Federal University of São Carlos (UFSCar), São Carlos, SP, Brazil.
authors: Vivian E
authors: VE
authors: Cornelio
authors: Department of Chemistry, Federal University of São Carlos (UFSCar), São Carlos, SP, Brazil.
authors: Mariele M
authors: MM
authors: Pedroso
authors: Department of Chemistry, Federal University of São Carlos (UFSCar), São Carlos, SP, Brazil.
authors: André S
authors: AS
authors: Afonso
authors: Department of Chemistry, Federal University of São Carlos (UFSCar), São Carlos, SP, Brazil.
authors: João B
authors: JB
authors: Fernandes
authors: Department of Chemistry, Federal University of São Carlos (UFSCar), São Carlos, SP, Brazil.
authors: M Fátima G F
authors: MF
authors: da Silva
authors: Department of Chemistry, Federal University of São Carlos (UFSCar), São Carlos, SP, Brazil. Electronic address: rcfaria@ufscar.br.
authors: Ronaldo C
authors: RC
authors: Faria
authors: Department of Chemistry, Federal University of São Carlos (UFSCar), São Carlos, SP, Brazil. Electronic address: dpcv@ufscar.br.
authors: Paulo C
authors: PC
authors: Vieira
mesh_terms: D001688:Biological Products / Q000032:analysis* / Q000494:pharmacology; D015374:Biosensing Techniques / Q000295:instrumentation* / Q000379:methods*; D002402:Cathepsin D / Q000037:antagonists & inhibitors / Q000378:metabolism; D006454:Hemoglobins / Q000032:analysis / Q000378:metabolism*; D006801:Humans; D006868:Hydrolysis / Q000187:drug effects; D011480:Protease Inhibitors / Q000032:analysis* / Q000494:pharmacology; D058525:Quartz Crystal Microbalance Techniques; D013329:Structure-Activity Relationship; D013997:Time Factors
chemical_list: D001688:Biological Products; D006454:Hemoglobins; D011480:Protease Inhibitors; D002402:Cathepsin D
keywords: Cathepsin D; Hemoglobin hydrolysis; Quartz crystal microbalance; Real-time monitoring