Development of a fast liquid chromatography/mass spectrometry screening method for angiotensin-converting enzyme (ACE) inhibitors in complex natural mixtures like snake venom.

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).

Isolation and characterization of two new Lys49 PLA2s with heparin neutralizing properties from Bothrops moojeni snake venom.

Among the proteins and peptides already characterized in Bothrops moojeni venom, two novel phospholipases A(2) (PLA(2)) have been purified and fully sequenced by ESI-MS/MS techniques. Both of them belong to the enzymatically non-active Lys49 variants of PLA(2). They consist of 122 amino acids and share a characteristic sequence in their C-terminal region composed of clusters of basic amino acids known to interact with heparin. Thus, as already established, heparin can be used as an antidote to antagonize some myotoxic PLA(2)s from venoms of Bothrops genus. The two PLA(2) variants were shown to interact in vitro with unfractionated heparin (UFH) and low molecular weight heparin (LMWH), neutralizing their anticoagulant properties. Although the influences of PLA(2)s from snake venoms on the blood coagulation system are known, their use to antagonize the anticoagulant effect of heparin in vitro or in vivo has never been proposed. These finding recommend diagnostic and therapeutic applications, which are currently investigated.

Platelet-active substances in the venom of Bothrops moojeni snake-a novel evaluation method using whole blood aggregometry.

The objective of the present study was an investigation of the crude Bothrops moojeni venom, aiming at the identification of new compounds with platelet-activating or -inhibiting activity. The venom was separated by gel filtration chromatography into 18 fractions, which were tested by means of whole blood aggregometry for their activities affecting the aggregation of blood platelets. In order to eliminate interferences caused by prothrombin activators or thrombin like-enzymes, which are frequently present in snake venoms, a test method for screening protein mixtures was developed. To avoid clotting of the blood samples, the thrombin inhibitor hirudin and the synthetic inhibitor of fibrin polymerization Pefabloc FG were applied. In the present study, a platelet aggregation activator with an activity resembling thrombocytin from B. atrox was identified in one of the examined venom fractions. In addition, a platelet antagonist-most likely a disintegrin-with broad inhibitory activity against aggregation triggered by collagen, adenosine diphosphate and thrombin receptor activating peptide, was identified.

Screening for proteolytic activities in snake venom by means of a multiplexing electrospray ionization mass spectrometry assay scheme.

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.

The potential of Bothrops moojeni venom in the field of hemostasis. Established use and new insights.

Early studies in the 1930s on the venom of South American Lancehead snakesofthe Bothrops genuslead to the discovery of compounds active in blood coagulation such as batroxobin and botrocetin. The scope of our investigations is to have a deeper look at the crude venom of B. moojeni using state-of-the-art proteomics methods, as well as newly developed bioassays screening for activities in the different fields of application. The proteomics techniques used up to now have included different chromatography methods, mass spectrometry, and bio-computing. The bioassays are focussed on enzymatic and other activities in the field of hemostasis and fibrinolysis. Besides the known activities several new and interesting ones have been found. They still need to be studied and confirmed in more specific supplementary assays.