ArticleMethods and protocols2025
An Optimized Protocol for Enzymatic Hypothiocyanous Acid Synthesis.
Article in Methods and protocols, 2025. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Not yet cited in PubMed.
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Abstract
Investigation of molecular mechanisms that underlie the toxicity of reactive oxidants requires the usage of reductionist cellular models, where laboratory cultures are treated by known doses of the target compounds in strictly controlled conditions. In recent years, much attention has been focused on hypothiocyanous acid (HOSCN), a pseudohypohalous acid that is one of the main products of chordata heme peroxidases. Due to its instability, HOSCN cannot be purchased and stored, so it has to be enzymatically synthesized before each experiment. For the first time, we systematically classified the published protocols for HOSCN synthesis, compared them by product yield, and found that the highest achievable concentration was about 1.9 mM. This value is not convenient for large-scale experiments with high cell density. Therefore, we developed an improved protocol for HOSCN preparation by optimizing reagent ratios, incubation times, and temperature. The current paper describes all steps from scratch, namely lactoperoxidase purification via a combination of cation exchange, hydrophobic interaction, and size exclusion chromatography, HOSCN synthesis from SCN
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