PURIFICATION AND CHARACTERIZATION OF PROLIXIN-S (NITROPHORIN-2), THE SALIVARY ANTICOAGULANT OF THE BLOODSUCKING BUG RHODNIUS-PROLIXUS

The salivary anticoagulant of the blood-sucking bug Rhodnius prolixus was purified to homogeneity using a protocol consisting of weak cation -exchange, DEAE, hydrophobic-interaction and octadecyl reverse-phase c hromatography, yielding a protein with the same N-terminal sequence as nitrophorin 2, one of the four NO haem protein carriers present in th e salivary glands of Rhodnius with a molecular mass of 19689 Da [D. Ch ampagne, R.H. Nussenzveig and J.M.C. Ribeiro, (1995) J. Biol. Chem. 27 0, in the press]. To exclude the possibility of the nitrophorin being a contaminant, another chromatographic protocol was performed, consist ing of chromatofocusing followed by strong-cation-exchange chromatogra phy. Again the anticoagulant was eluted with nitrophorin 2. Nitrophori n 2 inhibits coagulation Factor VIII-mediated activation of Factor X a nd accounts for all the anti-clotting activity observed in Rhodnius sa livary glands.