In vitro tracking of phospholipase A2 from snake venom conjugated with magic-sized quantum dots.

Phospholipases A2 represent a family of enzymes with important application in medicine. However, direct tracking is difficult due to the absence of a stable, effective and specific marker for these enzymes. Magic-sized quantum dots (MSQDs) are inorganic semiconducting nanocrystals with unique physical properties. They have the ability to conjugate to proteins, making them excellent markers for biological systems. In this work, we labelled phospholipase A2 from Bothrops alternatus snake venom with Cadmium selenide (CdSe)/cadmium sulphate (CdS) MSQDs-a biocompatible and luminescent probe-. Bioconjugation was confirmed using infrared spectra and fluorescence microscopy, which demonstrated that the CdSe/CdS MSQDs interact with phospholipase A2 without interfering with its activity. This probe may be an important tool for the elucidation of many biological mechanisms, because it allows the pathway of phospholipase A2 to be tracked from its entry through the plasma membrane until its incorporation into the nucleus of myoblasts.

Rapid purification of a new P-I class metalloproteinase from Bothrops moojeni venom with antiplatelet activity.

The present study aimed to evaluate the proteolytic and biological activities of a new metalloproteinase from B. moojeni venom. The purification of BmooMP α -II was carried out through two chromatographic steps (ion-exchange and affinity). BmooMP α -II is a monomeric protein with an apparent molecular mass of 22.5 kDa on SDS-PAGE 14% under nonreducing conditions. The N-terminal sequence (FSPRYIELVVVADHGMFTKYKSNLN) revealed homology with other snake venom metalloproteinases, mainly among P-I class. BmooMP α -II cleaves A α -chain of fibrinogen followed by B ß -chain, and does not show any effect on the γ -chain. Its optimum temperature and pH for the fibrinogenolytic activity were 30-50°C and pH 8, respectively. The inhibitory effects of EDTA and 1,10-phenantroline on the fibrinogenolytic activity suggest that BmooMP α -II is a metalloproteinase. This proteinase was devoid of haemorrhagic, coagulant, or anticoagulant activities. BmooMP α -II caused morphological alterations in liver, lung, kidney, and muscle of Swiss mice. The enzymatically active protein yet inhibited collagen, ADP, and ristocetin-induced platelet aggregation in a concentration-dependent manner. Our results suggest that BmooMP α -II contributes to the toxic effect of the envenomation and that more investigations to elucidate the mechanisms of inhibition of platelet aggregation may contribute to the studies of snake venom on thrombotic disorders.