Analysis of human beta-endorphin 28-31 (melanotropin potentiating factor) and analogues by high-performance liquid chromatography of their 9-fluorenylmethoxycarbonyl derivatives.
A method is described for the determination of sub-picomole amounts of Lys-Lys-Gly-Glu [the C-terminal tetrapeptide sequence of human beta-endorphin, referred to as melanotropin potentiating factor (MPF)], a putative neurotrophic agent. Attempts to raise antibodies to the peptide were not successful and we have therefore developed a method based on the fluorescence of its 9-fluorenylmethyl chloroformate (FMOC) derivative which provides a sensitivity comparable to that of radioimmunoassay. Standard solutions, cerebrospinal fluid or central nervous tissue extracts are first treated with FMOC-Cl. The resulting mixture of FMOC-peptides is then subjected to high-performance liquid chromatography (HPLC) and quantified using a fluorescence monitor. By this procedure, MPF and related peptides can be analysed from one sample in a single HPLC run. The method was also applied to determine the rate of release into a phosphate-buffered saline medium of a metabolically stable analogue of MPF from a slow-release formulation of the compound.
Lewis
J R
JR
Pain Research Institute, Pain Relief Foundation, Liverpool, UK.
Morley
J S
JS
Venn
R F
RF
D000595:Amino Acid Sequence; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D003692:Delayed-Action Preparations; D004151:Dipeptides / Q000008:administration & dosage / Q000032:analysis* / Q000134:cerebrospinal fluid; D004723:Endorphins; D005449:Fluorenes; D006801:Humans; D007202:Indicators and Reagents; D008297:Male; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010446:Peptide Fragments; D051381:Rats; D017208:Rats, Wistar; D013696:Temperature
D003692:Delayed-Action Preparations; D004151:Dipeptides; D004723:Endorphins; D005449:Fluorenes; D007202:Indicators and Reagents; D010446:Peptide Fragments; C050079:lysyl-lysyl-glycyl-glutamic acid; C054007:1-(9-fluorenyl)methyl chloroformate