通过反相高效液相色谱法测定弹性蛋白酶活性

Determination of elastase activity by reversed-phase high-performance liquid chromatography.

作者信息

Stein T A, Cohen J R, Mandell C, Wise L

机构信息

Department of Surgery, Long Island Jewish Medical Center, New Hyde Park, NY 11042.

出版信息

J Chromatogr. 1989 Jan 6;461:267-70. doi: 10.1016/s0021-9673(00)94292-4.

DOI:10.1016/s0021-9673(00)94292-4

PMID:2708474

Abstract

The action of elastase on elastin was measured by high-performance liquid chromatography with on-line post-column derivatization. After alkaline hydrolysis, a 20-ml sample was injected into a ODS-2 gel column. An elastin-specific dipeptide, Val-Pro, and the internal standard, Gly-Leu, were eluted by a linear gradient of 0 to 10% of 1-propanol in 50 mM heptafluorobutyrate (pH 3) at a flow-rate of 1 ml/min. The eluent was reacted with fluorescamine, and the fluorescent products were measured. Retention times for Val-Pro and Gly-Leu were 17.33 and 23.54 min. The peak areas of 0.2-16 micrograms of Val-Pro gave a straight-line plot. Elastase activity was constant from 6 to 24 h and was 0.95 +/- 0.02 (S.D.) micrograms/h. The method may be useful for the measurement of the elastolytic activity in some diseased tissues.

摘要

通过在线柱后衍生化的高效液相色谱法测定弹性蛋白酶对弹性蛋白的作用。碱性水解后，将20毫升样品注入ODS - 2凝胶柱。弹性蛋白特异性二肽Val - Pro和内标Gly - Leu在50 mM七氟丁酸（pH 3）中以0至10%的1 - 丙醇线性梯度洗脱，流速为1毫升/分钟。洗脱液与荧光胺反应，并测量荧光产物。Val - Pro和Gly - Leu的保留时间分别为17.33和23.54分钟。0.2 - 16微克Val - Pro的峰面积呈直线关系。弹性蛋白酶活性在6至24小时内保持恒定，为0.95±0.02（标准差）微克/小时。该方法可能有助于测量某些患病组织中的弹性蛋白分解活性。