A chemical method to isolate hypothalamic nonapeptides by coupling cyst(e)in with bimane.
Vasopressin, oxytocin as well as other active nonapeptides (vasotocin, etc) are difficult to isolate from tissues. Traditionally they were identified using cumbersome biological assays or immunoassays, commercially unavailable, and with some cross reactivity. Based on the fact that all these peptides have two Cysteines in their molecules we developed a simple, sensitive and specific method to detect them by HPLC after pre-column fluorescent derivatization with monobromobimane (mBBr). The peptides were separated on a Vydac C18 column after reduction with Tris (2-carboxyethyl) phosphine (TCEP) and derivatization with mBBr for 5 minutes in dark. Using this method we were able to detect specific peaks for arginine-, lysine-vasopressin, and vasotocin at levels as low as 10 pmol. The method can be used to detect other active peptides with cyst(e)ins in their molecule, as well.
Catrina
S B
SB
Department of Endocrinology II, University of Medicine and Pharmacy Carol Davila, Bucharest, Romania. s_catrina@hotmail.com
Coculescu
M
M
Andersson
M
M
D001127:Arginine Vasopressin / Q000302:isolation & purification; D001671:Biochemistry / Q000379:methods*; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000737:chemistry*; D002851:Chromatography, High Pressure Liquid; D003545:Cysteine / Q000737:chemistry*; D004220:Disulfides; D008236:Lypressin / Q000302:isolation & purification; D010455:Peptides / Q000737:chemistry* / Q000302:isolation & purification*; D010720:Phosphines / Q000494:pharmacology
D001127:Arginine Vasopressin; D019086:Bridged Bicyclo Compounds, Heterocyclic; D003545:Cysteine; D004220:Disulfides; D008236:Lypressin; D010455:Peptides; D010720:Phosphines; C021149:bimanes; C080938:tris(2-carboxyethyl)phosphine