Determination of elastase activity by reversed-phase high-performance liquid chromatography.
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.
Stein
T A
TA
Department of Surgery, Long Island Jewish Medical Center, New Hyde Park, NY 11042.
Cohen
J R
JR
Mandell
C
C
Wise
L
L
D002851:Chromatography, High Pressure Liquid; D004151:Dipeptides / Q000032:analysis; D004549:Elastin / Q000032:analysis; D010196:Pancreatic Elastase / Q000032:analysis* / Q000378:metabolism; D013050:Spectrometry, Fluorescence
D004151:Dipeptides; C034446:prolylvaline; D004549:Elastin; D010196:Pancreatic Elastase