Comparative in vitro degradation of the human hemorphin LVV‐H7 in mammalian plasma analysed by capillary zone electrophoresis and mass spectrometry

Abstract

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.

Kinetic studies on aminopeptidase M‐mediated degradation of human hemorphin LVV‐H7 and its N‐terminally truncated products

“…N -terminally truncated forms of LVV-H7 exhibit individual physiological properties with regard to receptor or enzyme affinities [8,10]. As recently shown in in vitro plasma stability studies, subsequent N -terminal truncation of LVV-H7 is catalyzed by AP-M [16] producing at least three proteolytic products as depicted in Figure 1. Therefore, we decided to investigate the enzymatic conversion by AP-M of both natural underivatized LVV-H7 and its metabolites acting as substrates for that exopeptidase, which potentially regulates in vivo activity of hemorphins as known for the opiate receptor-binding dynorphins [28].…”

“…Qualitative mass spectrometric detection of peptides from the incubation mixtures was carried out in the linear positive ion mode on a Voyager DE-Pro instrument (1.2 m flight tube, 337 nm laser, Applied Biosystems, Darmstadt, Germany) supported by the Biospectrometry Workstation 5.1 software for controlling and the Data Explorer 4.0 program for data analysis. Analyses were performed using the dried droplet technique [16].…”

“…In comprehensive plasma stability studies, we have recently shown that LVV-H7 is C-terminally truncated by ACE and additionally it is N -terminally hydrolyzed by the aminopeptidase M (AP-M, EC 3.4.11.2). This enzyme causes subsequent cleavage of at least three N -terminal amino acids producing the hemorphins VV-H7, V-H7, and H7, each of them exhibiting individual regulatory potency as discussed above [13,16] (Figure 1). AP-M, which is also named alanyl-AP [17], CD13 [18], membrane alanyl-AP or AP-N [19], is an ubiquitous O-and N -glycosylated [20] Zn 2+ -dependent metallo-exopeptidase with broad specificity.…”

“…So far, only a few studies have focused on hemorphin catabolism in, e.g. pure ACE solution [13], in rat brain and blood in vivo after microdialysis [2], or in vitro in subcellular fractions from rat brain [14], and different mammalian plasma [15,16]. In comprehensive plasma stability studies, we have recently shown that LVV-H7 is C-terminally truncated by ACE and additionally it is N -terminally hydrolyzed by the aminopeptidase M (AP-M, EC 3.4.11.2).…”

“…Furthermore, it is used as a marker for acute myeloid leukemia and plays a role in tumor invasion [18,26]. Only very little is known about hemorphin truncation by AP-M so far [16]. Therefore, we applied a novel validated CZE method [27] to analyze AP-M-mediated proteolysis of both the original underivatized LVV-H7 and its metabolites VV-H7, V-H7, and H7 to compare reaction velocities and substrate stabilities.…”