ISOLATION OF PHOSPHODIESTERASE FROM THE VENOM OF CROTALUS DURISSUS TERRIFICUS

FUSCO, L; FRANK TORRES-HUECO; HYSLOP S.; LEIVA L

Buenos Aires

Jornada; XVIII Jornadas Anuales Multidisciplinarias; 2016

Sociedad Argentina de Biología

Phosphodiesterases (PDEs) belong to a super-family of enzymes that have multiple roles in the metabolism of nucleotides. Snake venoms contain PDE (e.g. Trimeresurus stejnegeri, Daboia russelli russelli, Bothrops jararaca) but their function is poorly understood at the present. In this work a PDE was isolated from C.d.t venom (CDT-PDE) by ultrafiltration process in 50 KDa membranes. After that, proteins with a molecular mass more than 50KDa were concentrated and then purified by ion-exchange chromatographies (HiTrap Q-FF and SP XL). Homogeneity of the pool was verified by SDS-PAGE. Two bands around 100 KDa were analyzed by mass spectrometry (Q Exactive LC-MS/MS system) and the study of fragment peptides determined the presence of two fosfodiesterase isoforms (96 and 91 KDa). The activity of the CDT-PDEs on platelet aggregation was evaluated. When platelet rich plasma (350x103 /µl) was stimulated with 10 mM of ADP, platelet aggregation was completed by around 40%. However, in presence of 0.16µg of CDT-PDEs, the extent of aggregation reached at most 10%. This inhibitory effect is characteristic of other snake PDEs. This preliminary study will be expanded to characterize this enzyme of biomedical interest.