Isolation and purification of novel hirudins from the leech Hirudinaria manillensis by high-performance liquid chromatography.
The isolation and purification of novel hirudins from a crude extract of the leech Hirudinaria manillensis and their analytical characterization are reported. Initial purification by gel permeation chromatography on Sephadex G50 and anion-exchange chromatography on Q Sepharose fast-flow removed most contaminants and yielded a highly active extract. Two isohirudins (designated hirudin P6 and P18) were isolated and purified by successive reversed-phase high-performance liquid chromatography on silica-based stationary phases and anion-exchange chromatography on Mono Q. The final products were characterized by reversed-phase high-performance liquid chromatography, 252Cf plasma desorption time-of-flight mass spectrometry and capillary zone electrophoresis. The molecular masses determined by 252Cf plasma desorption mass spectrometry were 7416 dalton for hirudin P6 and 7199 dalton for hirudin P18.
Steiner
V
V
Pharmaceuticals Research Laboratories, Ciba-Geigy Ltd., Basel, Switzerland.
Knecht
R
R
Gruetter
M
M
Raschdorf
F
F
Gassmann
E
E
Maschler
R
R
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D004586:Electrophoresis; D004591:Electrophoresis, Polyacrylamide Gel; D006629:Hirudins / Q000302:isolation & purification*; D006801:Humans; D007865:Leeches / Q000378:metabolism*; D013058:Mass Spectrometry
D000596:Amino Acids; D006629:Hirudins