Serine proteinase inhibitors from eggs and larvae of tick Boophilus microplus: Purification and biochemical characterization

The present study describes the purification, characterization, and compari son of serine proteinase inhibitors during the development of egg and larva phases of the tick Boophilus microplus. Samples were collected of eggs bet ween the first day of hatching and the beginning of eclosion (defined as E1 , E2, and E3) and of larvae between the first day of eclosion and the infec tant phase (defined as L1, L2, and L3). Crude extracts of the samples (2.5% w/v in Tris-HCl buffer) were analyzed by SDS-PAGE, and showed three major protein bands of 42, 62, and 85 kDa, differing in intensity, from El to L3 samples. The total protein of the larva extracts was 34% less than that of the egg extracts, while no differences in active protein were detected. The apparent dissociation constant Ki determined for trypsin was 10-fold lower from E1 to L3 samples. Serine proteinase inhibitors from tick eggs and lar vae (BmTls) were purified on trypsin-Sepharose column and analyzed by SDS-P AGE. The results showed a slight difference in protein pattern, with a prot ein band of 20 kDa in the El and E2 samples which did not appear in the oth er samples. The K-i for neutrophil elastase was 10-fold lower in L3 than E1 . BmTI reverse-phase chromatography showed two and one major peaks in egg a nd larva samples, respectively. The N-terminal amino acid sequence of the L 3 main peak from a C8 column showed a mix of BmTls with the major sequence AVDFDKGCVPTADPGPCKG. Changes indicated by molecular weight and inhibition a ctivity suggest different roles for BmTIs during the development process.