Serra M A, Avilés F X, Giralt E, Cuchillo C M
Departament de Bioquímica i Biologia Molecular, Facultat de Ciències, Universitat Autonòma de Barcelona, Spain.
J Chromatogr. 1989 Sep 22;479(1):27-37. doi: 10.1016/s0021-9673(01)83314-8.
A reversed-phase high-performance liquid chromatographic (HPLC)-based method was developed to follow the time course of the hydrolytic action of pancreatic carboxypeptidase A on oligopeptide substrates of the general formula benzoyl(glycyl)n-L-Phe, n being in the range 1-5. The proposed procedure is sensitive at the nanomolar level of substrate, but also allows the kinetics of substrate hydrolysis to be investigated over a wide range of concentrations. The prior quenching of the carboxypeptidase activity and the use of isocratic conditions for the rapid separation of the substrates and their products (in less than 8 min for the slowest one), allow the automation of the procedure, which could become an easy and versatile alternative to the commonly used spectrophotometric methods. A comparative evaluation of the use of a spectrophotometric method to measure carboxypeptidase A activity on the same substrates indicated that the latter is not valid for long oligopeptides (n greater than or equal to 2) at concentrations higher than 5 mM owing to an interference of a physico-chemical nature. For benzoylglycyl-L-Phe (n = 1), the apparent kinetic parameters were calculated by means of the HPLC method and by a well established spectrophotometric procedure, and both yielded similar values.
开发了一种基于反相高效液相色谱（HPLC）的方法，用于跟踪胰羧肽酶A对通式为苯甲酰基（甘氨酰）n-L-苯丙氨酸的寡肽底物的水解作用随时间的变化过程，其中n在1至5的范围内。所提出的方法在底物的纳摩尔水平上具有灵敏度，但也允许在很宽的浓度范围内研究底物水解的动力学。羧肽酶活性的预先淬灭以及使用等度条件快速分离底物及其产物（最慢的产物在8分钟内），使得该方法能够自动化，这可能成为常用分光光度法的一种简便且通用的替代方法。对使用分光光度法测量羧肽酶A对相同底物的活性进行的比较评估表明，由于物理化学性质的干扰，后者对于浓度高于5 mM的长寡肽（n大于或等于2）无效。对于苯甲酰甘氨酰-L-苯丙氨酸（n = 1），通过HPLC方法和成熟的分光光度法程序计算了表观动力学参数，两者得出的值相似。
