Figure 1.
(A) Crovirin purification from Cvv venom using a reverse phase analytical C8 column, where the protein was eluted as peak 3. (B) SDS-PAGE analysis of peak 3 (lane B) containing the purified crovirin protein under reducing conditions. The gel was stained with Coomassie blue. Lane A, molecular weight markers. (C) MALDI-TOF mass spectrometry analyses of the intact protein yielded a molecular mass of 24,893.64 Da. The peaks of 12,424.36 and 12,477.62 Da correspond to doubly-charged (z = 2) cationic forms of crovirin.
Figure 2.
Alignment of crovirin peptide sequences (identified by MALDI-TOF-TOF) and homologous sequences from Calloselasma rhodostoma (gi:190195317), Crotalus viridis viridis (gi:190195319) and Gloydius blomhoffi (ablomin protein- gi:48428846) deposited in GenBank.
Bold letters highlight identical residues in the same position of all protein sequences.
Figure 3.
Cytotoxicity analyses of crovirin on LLC-MK2 cells (A) and peritoneal macrophages (B) by trypan blue cell viability and MTS assay, respectively.
No tested concentrations induced significant lost of cell viability in either cell type. Error bars represent standard deviation of the mean of 3 independent experiments.
Figure 4.
Crovirin effects on T. cruzi trypomastigotes (A) and intracellular amastigotes (B), L. amazonensis promastigotes (C) and intracellular amastigotes (D), and T. brucei rhodesiense PCF (procyclic form, in E) and BSF (bloodstream forms, in F).
T. cruzi trypomastigotes were treated with crovirin in RPMI medium with 10% FCS for 24 h only, since they do not survive in growth medium for longer and do not divide. T. brucei BSF and PCF and L. amazonensis promastigotes were treated with crovirin in complete growth media, for up to 72 h. Amastigotes of T. cruzi and Leishmania were treated with crovirin as dividing intracellular forms (infecting peritoneal mouse macrophages). Error bars represent standard deviation the mean of 3 independent experiments.
Table 1.
Crovirin activity towards medically important trypanosomes and Leishmania.