Equilibrium dialysis with HPLC detection to measure substrate binding affinity of a non-heme iron halogenase.
bioRxiv
; 2024 Apr 04.
Article
in En
| MEDLINE
| ID: mdl-38617253
ABSTRACT
Determination of substrate binding affinity (Kd) is critical to understanding enzyme function. An extensive number of methods have been developed and employed to study ligand/substrate binding, but the best approach depends greatly on the substrate and the enzyme in question. Below we describe how to measure the Kd of BesD, a non-heme iron halogenase, for its native substrate lysine using equilibrium dialysis with subsequent detection with High Performance Liquid Chromatography (HPLC). This method can be performed in anaerobic glove bag settings, requires readily available HPLC instrumentation for subsequent detection, and is adaptable to meet the needs of a variety of substrate affinity measurements.
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Collection:
01-internacional
Database:
MEDLINE
Language:
En
Journal:
BioRxiv
Year:
2024
Document type:
Article
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