采用具有活性连续监测系统的高效液相色谱法分离人唾液α-淀粉酶同工酶。
Separation of human salivary alpha-amylase isozymes by high-performance liquid chromatography with a continuous monitor system of the activity.
作者信息
Omichi K, Ikenaka T
机构信息
Department of Chemistry, Osaka University College of Science, Japan.
出版信息
Anal Biochem. 1988 Feb 1;168(2):332-6. doi: 10.1016/0003-2697(88)90326-0.
Human salivary alpha-amylase isozymes were rapidly separated from each other by high-performance liquid chromatography with a postcolumn assay. The eluate from the HPLC column was mixed continuously with an intramolecularly quenched fluorescent substrate, p-nitro-phenyl O-6-deoxy-6-[(2-pyridyl)amino]-alpha-D-glucopyranosyl-(1----4)-O-alpha-D- glucopyranosyl-(1----4)-O-alpha-D-glucopyranosyl-(1----4)-O-alpha-D- glucopyranosyl-(1----4)-alpha-D-glucopyranoside delivered by a pump. The mixture was incubated in a reaction coil, and the fluorescence intensity was continuously measured by a fluorescence detector. The assay was based on the marked increase in fluorescence with the enzymatic cleavage of the glycosidic bond of the substrate that links the fluorogenic and quenching moieties.
通过高效液相色谱柱后分析可快速分离人唾液α-淀粉酶同工酶。来自高效液相色谱柱的洗脱液与一种分子内淬灭荧光底物连续混合，该底物为对硝基苯基O-6-脱氧-6-[(2-吡啶基)氨基]-α-D-吡喃葡萄糖基-(1→4)-O-α-D-吡喃葡萄糖基-(1→4)-O-α-D-吡喃葡萄糖基-(1→4)-O-α-D-吡喃葡萄糖基-(1→4)-α-D-吡喃葡萄糖苷，由泵输送。混合物在反应盘管中孵育，荧光强度由荧光检测器连续测量。该分析基于底物糖苷键的酶促裂解导致荧光显著增加，该糖苷键连接着荧光基团和淬灭基团。
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