Yajima R, Chikuma T, Kato T
Research and Development Center, Dainabot Co., Chiba, Japan.
J Chromatogr B Biomed Appl. 1995 May 19;667(2):333-8. doi: 10.1016/0378-4347(95)00039-l.
A rapid and sensitive high-performance liquid chromatographic (HPLC)-fluorimetric assay method has been developed for the determination of carboxypeptidase H activity based on the measurement of N-(5-dimethyl-aminonaphthalene-1-sulfonyl)glycine (dansyl-Gly) formed enzymatically from dansyl-Gly-L-Lys or dansyl-Gly-L-Arg. Dansyl-Gly is eluted faster than the substrates with an N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (Hepes) buffer at pH 7.0 containing methanol, but eluted slower with an acidic buffer at pH 4.6. The new HPLC method separates the product and substrate in less than 5 min using an elution buffer at pH 7.0 containing 60% methanol. Using this method carboxypeptidase H activity has been detected in rat sciatic nerves. This HPLC method facilitates the assay of carboxypeptidase H activity in the enzyme samples from various tissues.
已开发出一种快速灵敏的高效液相色谱（HPLC）-荧光测定法，用于基于对由丹磺酰甘氨酸-L-赖氨酸或丹磺酰甘氨酸-L-精氨酸酶促形成的N-（5-二甲基氨基萘-1-磺酰基）甘氨酸（丹磺酰甘氨酸）的测量来测定羧肽酶H活性。在pH 7.0含甲醇的N-2-羟乙基哌嗪-N'-2-乙烷磺酸（Hepes）缓冲液中，丹磺酰甘氨酸的洗脱速度比底物快，但在pH 4.6的酸性缓冲液中洗脱较慢。新的HPLC方法使用含60%甲醇的pH 7.0洗脱缓冲液在不到5分钟内分离产物和底物。利用该方法已在大鼠坐骨神经中检测到羧肽酶H活性。这种HPLC方法便于测定来自各种组织的酶样品中的羧肽酶H活性。
