New approach for natural products screening by real-time monitoring of hemoglobin hydrolysis using quartz crystal microbalance

Autor(es)

Cornelio, Vivian E. ^[1] ; Pedroso, Mariele M. ^[1] ; Afonso, Andre S. ^[1] ; Fernandes, Joao B. ^[1] ; da Silva, M. Fatima G. F. ^[1] ; Faria, Ronaldo C. ^[1] ; Vieira, Paulo C. ^[1]

Afiliação do(s) autor(es)

^[1] Fed Univ Sao Carlos UFSCar, Dept Chem, Sao Carlos, SP - Brazil

• Tipo de documento: Artigo Científico
• Fonte: Analytica Chimica Acta; v. 862, p. 86-93, MAR 3 2015.
• Citações Web of Science: 5

Resumo

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. (C) 2015 Elsevier B.V. All rights reserved. (AU)

• Processo FAPESP: 09/17538-8 - Estudo de novas metodologias para realização de ensaios com a catepsina D na busca de inibidores
• Beneficiário: Vivian Estevam Cornélio
• Modalidade de apoio: Bolsas no Brasil - Doutorado Direto