High-performance affinity monolith chromatography for chiral separation and determination of enzyme kinetic constants |
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Authors: | Chunhe Yao Li Qi Juan Qiao Haizhi Zhang Fuyi Wang Gengliang Yang |
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Institution: | a Beijing National Laboratory for Molecular Sciences, Key Laboratory of Analytical Chemistry for Living Biosystems, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, China b Graduate School, Chinese Academy of Sciences, Beijing 100049, China c College of Pharmacy, Hebei University, Baoding 071002, China |
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Abstract: | A new kind of immobilized human serum albumin (HSA) column was developed by using the sub-micron skeletal polymer monolith based on poly(glycidyl methacrylate-co-ethylene glycol dimethacrylate) poly(GMA-EDMA)] as the support of high-performance affinity chromatography. Using the epoxide functional groups presented in GMA, the HSA immobilization procedure was performed by two different means. The affinity columns were successfully adopted for the chiral separation of d,l-amino acids (AAs). Then this method was shown to be applicable to the quantitative analysis of d-tryptophan, with a linear range between 12.0 μM and 979.0 μM, and a correlation coefficient above 0.99. Furthermore, it was used for the analysis of urine sample. This assay is demonstrated to be facile and relatively rapid. So it allows us to measure the enzyme catalytic activity in the incubation of d,l-AAs with d-AA oxidase and to study the kinetics of the enzyme reaction. It implied that the affinity monolithic columns can be a useful tool for studying DAAO enzyme reaction and investigating the potential enzyme mechanism requirement among chiral conversion. |
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Keywords: | High-performance affinity chromatography Human serum albumin Enzyme kinetic study Chiral separation Monolithic column |
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