Separation of alkylaminonaphthylenesulfonyl peptides and amin acids by high-performance liquid chromatography. Methods for measuring melanotropin inhibiting factor breakdown.
N,N-Dimethyl diethyl, dipropyl, dibutyl, and N-monoisopropylaminoaphthylenesulfonyl derivatives of melanotropin inhibiting factor (MIF) and its metabolites were prepared, and their chromatographic behavior was investigated with thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC), using five solvent systems on polyamide layers and ten solvent systems on muBondapak C18 and muBondapak phenyl columns. A mixture of MIF and its metabolites derivatized with Dns chloride was adequately resolved by two-dimensional chromatography on polyamide layer with solvent systems, formic acid-water (3:97) and benzene-acetic acid (9:1). Bns-MIF and its metabolites were separated with muBondapak C18 column with the solvent system acetonitrile-0.01 M sodium sulphate buffer, pH 7 (50:50). They were separated into five groups: Gly and Bns acid; Pro-Leu, Leu-Gly and Leu; Pro; Gly-NH2; and MIF. The alkylaminonaphthylenesulfonyl derivates had strong fluorescence, which permitted their detection at the level of 10(-11) to 10(-9) mol. Dns-MIF and its derivatives had the lowest detectable amounts. HPLC with the aid of the Dns derivation is reliable and fast, and is the preferable method for study of neuropeptide breakdown.
D000476:Alkanesulfonates / Q000032:analysis; D000596:Amino Acids / Q000032:analysis*; D000818:Animals; D001921:Brain / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D009075:MSH Release-Inhibiting Hormone / Q000032:analysis / Q000378:metabolism*; D010455:Peptides / Q000032:analysis
D000476:Alkanesulfonates; D000596:Amino Acids; D010455:Peptides; D009075:MSH Release-Inhibiting Hormone