Equilibrium dialysis with HPLC detection to measure substrate binding affinity of a non-heme iron halogenase.

Smithwick, Elizabeth R; Bhagi-Damodaran, Ambika; Damodaran, Anoop Rama

Smithwick, Elizabeth R; Bhagi-Damodaran, Ambika; Damodaran, Anoop Rama.

Afiliación

Smithwick ER; Department of Chemistry, University of Minnesota -Twin Cities, 207 Pleasant St. SE. Minneapolis, MN 55455.
Bhagi-Damodaran A; Department of Chemistry, University of Minnesota -Twin Cities, 207 Pleasant St. SE. Minneapolis, MN 55455.
Damodaran AR; Department of Chemistry, University of Minnesota -Twin Cities, 207 Pleasant St. SE. Minneapolis, MN 55455.

bioRxiv ; 2024 Apr 04.

Article en En | MEDLINE | ID: mdl-38617253

ABSTRACT

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.

Palabras clave

AQC derivatization; amino acid detection; biocatalysts; equilibrium dialysis; non-heme iron halogenases

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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Idioma: En Revista: BioRxiv Año: 2024 Tipo del documento: Article Pais de publicación: Estados Unidos

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