Cautionary Tale of Using Tris(alkyl)phosphine Reducing Agents with NAD⁺-Dependent Enzymes.

ABSTRACT: Protein biochemistry protocols typically include disulfide bond reducing agents to guard against unwanted thiol oxidation and protein aggregation. Commonly used disulfide bond reducing agents include dithiothreitol, β-mercaptoethanol, glutathione, and the tris(alkyl)phosphine compounds tris(2-carboxyethyl)phosphine (TCEP) and tris(3-hydroxypropyl)phosphine (THPP). While studying the catalytic activity of the NAD(P)H-dependent enzyme Δ¹-pyrroline-5-carboxylate reductase, we unexpectedly observed a rapid non-enzymatic chemical reaction between NAD⁺ and the reducing agents TCEP and THPP. The product of the reaction exhibits a maximum ultraviolet absorbance peak at 334 nm and forms with an apparent association rate constant of 231-491 M^-1 s^-1. The re

SUBMITTER: Patel SM 

PROVIDER: S-EPMC8528484 | biostudies-literature | 2020 Sep

REPOSITORIES: biostudies-literature