RESUMO
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.
RESUMO
OBJECTIVE: To establish the quantification method for free amino acids including threonine, alanine, lysine and tyrosine in the inner part of Poria cocos. METHODS: AQC (6-quinolyl-N-hydroxy-succinimidyl-carboxylate) was used as deriving reagent, and pre-column derivatization combined with ultra-high performance chromatography was applied to quantify threonine, alanine, lysine and tyrosine in 24 batches of the inner part of Poria cocos from Yunnan, Hubei and Anhui provinces. The experimental data were analyzed by partial least squares discriminant analysis (PLS-DA) using Simca-p+11 software. RESULTS: Alanine existed in every batch of sample, and its content ranged from 16.97-69.92 μg·g-1 in samples collected from different origins. Threonine, lysine and tyrosine were not found in some samples. The PLS-DA result showed that the samples from Yunnan and Anhui provinces clustered well. While the amino acid contents in the samples from Hubei province varied greatly. CONCLUSION: AQC combined with pre-column derivatization is a convenient, rapid and sensitive method to quantify the free amino acids in Poria.
