High-performance liquid chromatographic method for the determination of epsilon-(gamma-glutamyl)lysine and mono- and bis-gamma-glutamyl derivatives of putrescine and spermidine.
A sensitive, simple, and rapid high-performance liquid chromatographic (HPLC) method is reported for the determination of epsilon-(gamma-glutamyl)lysine and certain gamma-glutamylpolyamines in selected fractions from ion-exchange chromatograms of protein digests. The method involves pre-column derivatization of the gamma-glutamylamine conjugates with o-phthalaldehyde, linear-gradient reversed-phase HPLC separation, and fluorimetric detection. The gradient used was designed to provide a means of avoiding a desalting step, while maintaining proper chromatographic performance. gamma-Glutamylamines in amounts from 0.1 to 1 nmol display linear concentration-response relationships. The detection limits are approximately 10 and 200 pmol per mg of protein for the gamma-glutamylpolyamines and for epsilon-(gamma-glutamyl)lysine, respectively. The use of the method is exemplified by an analysis of the epidermal cell envelope from the skin of a newborn mouse.
Beninati
S
S
Laboratory of Oral Biology and Physiology, National Institute of Dental Research, Bethesda, MD 20892.
Martinet
N
N
Folk
J E
JE
D000818:Animals; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D004151:Dipeptides / Q000302:isolation & purification*; D006801:Humans; D051379:Mice; D011700:Putrescine / Q000031:analogs & derivatives* / Q000302:isolation & purification; D013095:Spermidine / Q000031:analogs & derivatives* / Q000302:isolation & purification
D004151:Dipeptides; C001299:epsilon-(gamma-glutamyl)-lysine; C011830:glutamylputrescine; C055363:N(1)-(gamma-glutamyl)spermidine; D013095:Spermidine; D011700:Putrescine