| Full text | |
| Author(s): |
Lima, Juliana M.
[1]
;
Seidl, Claudia
[1]
;
Cunha, Elise M. F.
[1, 2]
;
Oliveira, Arthur H. C.
[1]
;
Cardoso, Carmen L.
[1]
Total Authors: 5
|
| Affiliation: | [1] Univ Sao Paulo, Dept Quim, Grp Cromatog Bioafinidade & Prod Nat, Fac Filosofia Ciencias & Letras Ribeirao Preto, BR-14040900 Ribeirao Preto, SP - Brazil
[2] Inst Fed Educ Ciencia & Tecnol Rondonia, Campus Ji Parana, BR-76900000 Ji Parana, RO - Brazil
Total Affiliations: 2
|
| Document type: | Journal article |
| Source: | Journal of the Brazilian Chemical Society; v. 30, n. 11, SI, p. 2308-2317, NOV 2019. |
| Web of Science Citations: | 0 |
| Abstract | |
We describe an on-flow zonal affinity-based chromatography assay to screen ligands for the nucleoside diphosphate kinase B enzyme (NME2) from Homo sapiens. For the first time, we have covalently immobilized NME2 on the surface of an open fused silica capillary reactor (NME2-ICER) and placed the reactor before the analytical column, which resulted in a two-dimensional liquid chromatography-based system. We evaluated the pH effect on immobilized NME2 activity and carried out steady-state kinetic studies to compare free and immobilized NME2. Steady-state kinetic studies with the substrates adenosine 5'-triphosphate di(tris) salt dihydrate (ATP) and guanosine 5'-diphosphate sodium salt (GDP) resulted in apparent Michaelis-Menten constant values of 1136 and 713 mmol L-1, respectively. The ping-pong catalysis mechanism and substrate specificity were preserved after NME2 immobilization. By employing a reference inhibitor, (-)-epicatechin gallate (ECG), we verified the potential application of this method in NME2 ligand screening and NME2 inhibitor identification. The half maximum inhibitory concentration (IC50) for ECG was 161.3 +/- 1.0 mu mol L-1. (AU) | |
| FAPESP's process: | 14/50249-8 - Green chemistry: sustainable synthetic methods employing benign solvents, safer reagents, and bio-renewable feedstock |
| Grantee: | Arlene Gonçalves Corrêa |
| Support Opportunities: | Research Grants - Research Centers in Engineering Program |
| FAPESP's process: | 13/01710-1 - Enzyme ligand: new models of screening |
| Grantee: | Quezia Bezerra Cass |
| Support Opportunities: | Research Projects - Thematic Grants |
| FAPESP's process: | 14/06907-0 - Immobilization nucleoside diphosphate kinase (NDK) enzyme Leishmania major: the study of conditions for the application in the screening of ligands |
| Grantee: | Juliana Maria de Lima |
| Support Opportunities: | Scholarships in Brazil - Doctorate (Direct) |
| FAPESP's process: | 14/11640-3 - LC-MS / 2D methods for screening ligands for evaluating enzyme inhibitory activity and identification of active compounds in crude extracts |
| Grantee: | Cláudia Seidl |
| Support Opportunities: | Scholarships in Brazil - Post-Doctoral |