Isolation and characterization of a novel metalloprotease inhibitor from Bothrops alternatus snake serum.
Resistance of snakes and some other animals to snake envenomation has been attributed to soluble factors present in their tissues. Here we report the isolation of a novel metalloprotease inhibitor from Bothrops alternatus snake serum (named BaltMPI) with high purity, using a four-step chromatographic method. BaltMPI has molecular weights of 60.5 and 42.4kDa, as determined by SDS-PAGE and mass spectrometry, respectively, and pI=5.27. The first 60 amino acids from the N-terminal region of BaltMPI, determined by Edman's degradation, showed high homology (97%) with the snake venom metalloprotease inhibitor (SVMPI) BJ46a and other SVMPIs (78-82%). The chromatographic fractions and purified BaltMPI exhibited anti-hemorrhagic activity against Batroxase and BjussuMP-I. BaltMPI was stable over wide ranges of pH (1, 5, 8, and 9) and temperature (-80, -20, 4, 60, and 100°C), and suppressed the fibrinogenolytic, fibrinolytic, and azocaseinolytic activities of Batroxase. BaltMPI specifically inhibited the activity of metalloproteases, without affecting the activity of serine proteases. Together, our results suggest that BaltMPI and other SVMPIs are promising molecules for the treatment of snake envenomation, in particular that caused by Bothrops sp.
School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo (FCFRP-USP), Ribeirão Preto, SP, Brazil.
Tatiana Z
TZ
Palacio
Chemistry Institute (IQ), UNESP - Universidade Estadual Paulista, Araraquara, SP, Brazil.
Norival A
NA
Santos-Filho
Protein Chemistry Center, Department of Cell and Molecular Biology and Pathogenic Bioagents, Ribeirão Preto Medical School, University of São Paulo (FMRP-USP), Ribeirão Preto, SP, Brazil.
José Cesar
JC
Rosa
Center for the Study of Venoms and Venomous Animals (CEVAP), UNESP - Universidade Estadual Paulista, Botucatu, SP, Brazil; Botucatu Medical School (FMB), UNESP - Universidade Estadual Paulista, Botucatu, SP, Brazil.
Rui S
RS
Ferreira
Center for the Study of Venoms and Venomous Animals (CEVAP), UNESP - Universidade Estadual Paulista, Botucatu, SP, Brazil; Botucatu Medical School (FMB), UNESP - Universidade Estadual Paulista, Botucatu, SP, Brazil.
Benedito
B
Barraviera
School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo (FCFRP-USP), Ribeirão Preto, SP, Brazil. Electronic address: suvilela@usp.br.
Suely V
SV
Sampaio
D000595:Amino Acid Sequence; D000818:Animals; D017837:Bothrops / Q000097:blood*; D002364:Caseins / Q000378:metabolism; D005337:Fibrin / Q000378:metabolism; D005340:Fibrinogen / Q000378:metabolism; D006470:Hemorrhage / Q000188:drug therapy; D008666:Metalloendopeptidases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D051379:Mice; D011480:Protease Inhibitors / Q000097:blood / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology*; D059748:Proteolysis / Q000187:drug effects
D002364:Caseins; D011480:Protease Inhibitors; C026018:azocasein; D005337:Fibrin; D005340:Fibrinogen; D008666:Metalloendopeptidases
Anti-hemorrhagic activity; BaltMPI; Bothrops alternatus; Metalloprotease inhibitor; Snake serum; Snake venom metalloprotease