Liquid chromatography studies on the enzymatic degradation of luteinizing hormone-releasing hormone analogues with off-line identification by mass spectrometry.
Several agonists of luteinizing hormone-releasing hormone (LHRH) are currently used for different therapeutic purposes, but relatively little is known about their metabolic fate after administration. This paper describes the application of high-performance liquid chromatography combined with off-line fast atom bombardment mass spectrometry to identify the degradation products resulting from the incubation of LHRH analogues with proteolytic enzymes. Three analogues, containing a psi(E,CH = CH) pseudo-peptide bond were synthesized and afforded to the assay to determine the resistance against alpha-chymotrypsin and subtilisin: [Tyr5 psi(E,CH = CH)Gly6]LHRH, [Gly6 psi(E,CH = CH)D,L-Leu7]LHRH and [Pro9 psi(E,CH = CH)Gly10 ]LHRH. The pattern of peptide metabolites identified by this method indicates that alpha-chymotrypsin degrades LHRH analogues at the Trp3-Ser4 and Tyr5-Gly6 bond, while subtilisin hydrolyzes only the Tyr5-Gly6 linkage. The results also indicate a possible stabilization of native amide bonds against enzymatic degradation by neighbouring psi(E, CH = CH) modifications.
Péter
A
A
Department of Inorganic and Analytical Chemistry, Attila József University, Szeged, Hungary.
Devadder
S
S
Laus
G
G
Tourwé
D
D
D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D002918:Chymotrypsin / Q000737:chemistry; D007987:Gonadotropin-Releasing Hormone / Q000032:analysis* / Q000737:chemistry; D007202:Indicators and Reagents; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000302:isolation & purification; D016339:Spectrometry, Mass, Fast Atom Bombardment; D013056:Spectrophotometry, Ultraviolet; D013381:Subtilisins / Q000737:chemistry
D007202:Indicators and Reagents; D010455:Peptides; D007987:Gonadotropin-Releasing Hormone; D013381:Subtilisins; D002918:Chymotrypsin