Purification of radioiodinated photoactivable glucagon by isocratic high-performance liquid chromatography.
2-Nitro-4-azidophenylsulphenyl-glucagon, a specific photoaffinity label for glucagon receptors, was synthesized and radioactively labelled with 125I. The radio-labelled peptide was purified from the reaction mixture by high-performance liquid chromatography in one step by isocratic elution from a C18 column with 20.4% n-propanol in 10 mM phosphate buffer (pH 2.5) as eluent. This glucagon derivative can be used to attach a label specifically to the glucagon receptor. The binding ability of the photoaffinity derivative was tested on isolated intact rat hepatocytes. Compared with a Kd of 3 nM for unmodified monoiodinated glucagon, the Kd value of the photoaffinity labelled monoiodinated glucagon tracer was 7 nM.
D000818:Animals; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D002918:Chymotrypsin; D004355:Drug Stability; D005934:Glucagon / Q000302:isolation & purification*; D006868:Hydrolysis; D007457:Iodine Radioisotopes; D008099:Liver / Q000378:metabolism; D010777:Photochemistry; D011869:Radioligand Assay; D051381:Rats; D014357:Trypsin
D007457:Iodine Radioisotopes; D005934:Glucagon; D002918:Chymotrypsin; D014357:Trypsin