Comparative in vitro degradation of the human hemorphin LVV-H7 in mammalian plasma analysed by capillary zone electrophoresis and mass spectrometry.
The human hemorphin LVV-H7 (L32VVYPWTQRF41) is a hemoglobin-beta, -gamma, -delta or -epsilon chain derived cationic decapeptide of the micro-opioid receptor binding family. It exhibits potential pharmacological value relevant, for example, for blood pressure regulation, learning performance and Alzheimer's disease. The regulatory potency is strictly dependent on the length of the amino acid sequence which is sensitive towards proteinases from tissues and plasma. To analyse LVV-H7 in vitro degradation in mammalian plasma, a novel multi-component quantitative capillary zone electrophoretic (CZE) procedure was applied, combined with qualitative metabolite profiling by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). In all types of plasma, LVV-H7 was N-terminally truncated generating four metabolites (M1-M4) with an intact C-terminus: M1 (V33VYPWTQRF41), M2 (V34YPWTQRF41), M3 (Y35PWTQRF41) and M4 (W37TQRF41). In EDTA plasma these degradation products were detected exclusively, whereas in citrate and heparin plasma four further metabolites appeared resulting from additional C-terminal cleavage of the dipeptide R40F41: M5 (L32VVYPWTQ39), M6 (V33VYPWTQ39), M7 (V34YPWTQ39) and M8 (Y35PWTQ39). In the presence of selective proteinase inhibitors aminopeptidase M and angiotensin-converting enzyme (for N- and C-terminal truncation, respectively) were identified as plasma enzymes responsible for hemorphin degradation. Furthermore, striking inter-mammalian species distinctions were detected revealing strongly differing degradation velocities but similar metabolite patterns.
John
Harald
H
IPF PharmaCeuticals GmbH, Feodor-Lynen-Str. 31, Hannover, Germany. HaraldJohn@bundeswehr.org
Schulz
Stefanie
S
Forssmann
Wolf-Georg
WG
D000818:Animals; D000925:Anticoagulants / Q000494:pharmacology; D001711:Biotransformation; D018826:CD13 Antigens / Q000378:metabolism; D053801:Capillary Electrochromatography; D002951:Citrates / Q000097:blood; D004492:Edetic Acid / Q000494:pharmacology; D006454:Hemoglobins; D006493:Heparin / Q000494:pharmacology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D013058:Mass Spectrometry; D010446:Peptide Fragments / Q000097:blood*; D012015:Reference Standards; D013045:Species Specificity; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013056:Spectrophotometry, Ultraviolet
D000925:Anticoagulants; D002951:Citrates; D006454:Hemoglobins; D010446:Peptide Fragments; C074309:LVV-hemorphin-7; D006493:Heparin; D004492:Edetic Acid; D018826:CD13 Antigens