A multiplexed mass spectrometry based assay scheme for the simultaneous determination of five different substrate/product pairs was developed as a tool for screening of proteolytic activities in snake venom fractions from Bothrops moojeni. The assay scheme was employed in the functional characterization of eight model proteases. Time-resolved reaction profiles were generated and the relative reaction progress at each time point was determined. These were used to semi-quantitatively sort the catalytic activities of each enzyme towards the respective substrates into six classes. The resulting activity pattern served as an activity fingerprint for each enzyme. The multiplex assay scheme was then applied to a screening for proteolytic activities in fractions of the pre-separated venom from B. moojeni. Activity patterns of each fraction were generated and used to sort the fractions into three different categories of activity. By comparison of the fingerprint activity patterns of the venom fractions and the model enzymes, a compound with proteolytic properties similar to activated protein C was detected.
A new robust high-performance liquid chromatography/electrospray ionization mass spectrometry (HPLC/ESI-MS)-based screening method for angiotensin-converting enzyme (ACE)-inhibiting substances in crude samples is described. The ACE assay is carried out in a typical offline setup by incubation of the samples with ACE and angiotensin I (AI), followed by stopping the reaction with acetonitrile containing val(5)-AI serving as internal standard (I.S.). AI and the product angiotensin II (AII) are extracted from the incubation mixture by turbulent-flow chromatography (TFC) applied in backflush mode as online solid-phase extraction and are directly quantified by ESI(+)-MS. The presence of ACE inhibitors (ACEi) is detected by an increase in AI signal intensity and a corresponding decrease of AII signal, as compared to the blank assay. The overall time of analysis of the TFC/ESI-MS method was 5 min, thus making the described setup suitable for a rapid screening method. The assay was validated using a known ACE inhibitor and the IC(50) values found were in good accordance with a common HPLC/UV method and literature data. The method was successfully applied for the screening of size-exclusion chromatography fractions of the venom of the pitviper Bothrops moojeni. Three of 18 analyzed fractions inhibited ACE, due to peptides present as components of this snake venom. These compounds were extracted from the two most-active fractions by means of TFC and isolated by means of HPLC. Three peptides with ACE inhibitory activity were characterized and their structures were elucidated with ESI-MS/MS-based de novo sequencing to be ZKWPPGKVPP, ZKWPRPGPEIPP and ZNWPRPGPEIPP, respectively (Z = pyroglutamic acid).
An electrospray ionization/mass spectrometry (ESI/MS)-based assay for the determination of acetylcholinesterase (AChE)-inhibiting activity in snake venom was developed. It allows the direct monitoring of the natural AChE substrate acetylcholine (AC) and the respective product choline. The assay scheme was employed in the screening for neurotoxic activity in fractions of the venom of Bothrops moojeni. AChE inhibition was assessed in two fractions. As a positive control, the established AChE inhibitor 1,5-bis(4-allyldimethylammoniumphenyl)pentan-3-one dibromide (BW284c51) was used, a dose-response curve for this compound was generated and the IC 50 value for the inhibitor was determined to be 1.60 ± 0.09 × 10 -9 mol L -1 . The dose-response curve was used as "calibration function" for the venom inhibition activity, resulting in BW284c51-equivalent concentrations of 1.76 × 10 -9 mol L -1 and 1.07 × 10 -9 mol L -1 for the two fractions containing activity. The ESI/MS-based assay scheme was validated using the established Ellman reaction. The data obtained using both methods were found to be in good agreement. The ESI/MS-based assay scheme is therefore an attractive alternative to the standard colorimetric assay.
Introduction: With progressing ageing human sebocytes reduce lipid production. However, the influence of certain aging mechanisms on sebaceous lipid synthesis as well as ways to influence the latter is not fully identified. Certain lipids act as ligands of nuclear receptors such as PPAR. Phospholipase (PLA2) catalyzes the hydrolysis of the sn‐2 fatty acyl bond of phospholipids to yield free fatty acid and lysophospholipid. It has been hypothesized that PPAR may be activated by hydrolysis products of phospholipids and also by eicosanoids obtained through PLA2 activity. Materials and Methods: A method to quantify sebaceous lipid synthesis of SZ95 sebocytes in vitro was established and the cells were treated by snake venom Bothrops moojeni gel filtration fractions (Botmo GF). Botmo GF fractions were further purified by RP‐HPLC, and a fraction with PLA2 activity (Botmo GF11‐117) and a fraction without enzymatic activity (Botmo GF11‐101) were identified and additionally tested. Results: Botmo GF fractions increased lipogenesis in SZ95 sebocytes without inducing apparent toxic or apoptotic effects. Botmo GF11‐101 (1 μg/ml) enhanced neutral lipid synthesis by up to 170% and polar lipid synthesis by up to 120%. The enzymatically active PLA2 Botmo GF11‐117 (1 μg/ml) increased synthesis of neutral lipids by up to 200%, and polar lipids by up to 120% compared to untreated SZ95 sebocytes. Conclusion: PLA2 activation or suppression could be important for human sebaceous lipogenesis. PLA2 modifiers may be attractive for skin lipid research and pharmacological/cosmetic products.
The scientific background for this work is based on the fact that sebocytes reduce lipid production during the skin aging process. The relationships between aging effects and lipid reduction on a molecular level and ways to influence them are not yet fully identified. For this reason we investigated lipid stimulation in SZ95 sebocytes by Bothrops moojeni snake venom gel filtration fractions (Botmo GF). Several Botmo GFs increased lipid synthesis in SZ95 sebocytes without apparent toxic or apoptotic effects in applied concentrations. Botmo GF 11 was further purified by RP-HPLC. Fraction Botmo GF 11-117 showed phospholipase A 2 (PLA 2 ) activity, whereas fraction Botmo GF 11-101 did not. At a concentration of 1 μg/ml Botmo GF 11-101 enhanced neutral lipid synthesis by 150% and Botmo GF 11-117 by 310% compared to untreated SZ95 sebocytes. It was supposed that sebum production is stimulated via a PPAR agonistic mechanism of free fatty acids that were released by PLA 2 activity. However, the present data surprisingly indicate that lipid synthesis stimulation by Botmo GF 11-101 and Botmo GF 11-117 is independent of PLA 2 enzymatic activity in Botmo GF 11 subfractions. Thus, it is suspected that both Bothrops moojeni fractions act via different mechanisms on lipid stimulation in SZ95 sebocytes.Although the exact mode of actions are not yet identified, the Botmo GF 11-101 and Botmo GF 11-117 might be interesting tools for the investigation of sebocyte lipogenesis and may be helpful to the development of therapeutic concepts for the treatment of age-related skin dryness.
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