title: Isolation and biochemical characterization of a γ-type phospholipase A2 inhibitor from Crotalus durissus collilineatus snake serum.
abstract: In the present work, we describe the isolation and partial structural and biochemical characterization of the first phospholipase A2 inhibitor (γPLI) from Crotalus durissus collilineatus (Cdc) snake serum. Initially, the Cdc serum was subjected to a Q-Sepharose ion exchange column, producing six peaks at 280 nm absorbance (Q1-Q6). Subsequently, Q4 fraction was submitted to affinity chromatography with immobilized PLA2 BnSP-7, a step that resulted in two fractions (NHS-1 and NHS-2). The latter contained the inhibitor, denominated γCdcPLI. The molecular mass of γCdcPLI, determined by Matrix-Assisted Laser Desorption Ionization Time-of-Flight (MALDI-TOF), was 22,340 Da. Partial sequences obtained by Edman degradation and by mass spectrometry (MALDI-TOF/TOF), showed similarity, as expected, to other related inhibitors. Circular dichroism (CD) analysis showed the presence of approximately 22% alpha helices and 29% beta sheets in the protein secondary structure. Additionally, CD studies also indicated no significant changes in the secondary structure of γCdcPLI when it is complexed to BpPLA2-TXI. On the other hand, dynamic light scattering (DLS) assays showed a temperature-dependent oligomerization behavior for this inhibitor. Biochemical analyses showed γCdcPLI was able to inhibit the enzymatic, cytotoxic and myotoxic activities of PLA2s. Structural and functional studies performed on this inhibitor may elucidate the action mechanisms of PLA2 inhibitors. In addition, we hope this study may contribute to investigating the potential use of these inhibitors for the treatment of snakebite or inflammatory diseases in which PLA2s may be involved.
authors: Instituto de Genética e Bioquímica, Universidade Federal de Uberlândia, UFU, Uberlândia, MG, Brazil.
authors: Sarah Natalie Cirilo
authors: SN
authors: Gimenes
authors: Instituto de Genética e Bioquímica, Universidade Federal de Uberlândia, UFU, Uberlândia, MG, Brazil.
authors: Francis Barbosa
authors: FB
authors: Ferreira
authors: Instituto de Genética e Bioquímica, Universidade Federal de Uberlândia, UFU, Uberlândia, MG, Brazil.
authors: Ana Carolina Portella
authors: AC
authors: Silveira
authors: Instituto de Genética e Bioquímica, Universidade Federal de Uberlândia, UFU, Uberlândia, MG, Brazil; INCT, Instituto Nacional de Ciência e Tecnologia em Nano-Biofarmacêutica, Brazil.
authors: Renata Santos
authors: RS
authors: Rodrigues
authors: Instituto de Genética e Bioquímica, Universidade Federal de Uberlândia, UFU, Uberlândia, MG, Brazil.
authors: Kelly Aparecida Geraldo
authors: KA
authors: Yoneyama
authors: Departamento de Física e Biofísica, Instituto de Biociências, Universidade Estadual Paulista, UNESP, Botucatu, SP, Brazil.
authors: Juliana
authors: J
authors: Izabel Dos Santos
authors: Departamento de Física e Biofísica, Instituto de Biociências, Universidade Estadual Paulista, UNESP, Botucatu, SP, Brazil.
authors: Marcos Roberto de Mattos
authors: MR
authors: Fontes
authors: Instituto de Biologia, Universidade Federal de Uberlândia, UFU, Uberlândia, MG, Brazil.
authors: Vera Lúcia
authors: VL
authors: de Campos Brites
authors: Faculdade de Medicina Veterinária, Universidade Federal de Uberlândia, UFU, Uberlândia, MG, Brazil.
authors: André Luiz Quagliatto
authors: AL
authors: Santos
authors: Fundação Ezequiel Dias, FUNED, Belo Horizonte, MG, Brazil.
authors: Márcia Helena
authors: MH
authors: Borges
authors: Instituto de Genética e Bioquímica, Universidade Federal de Uberlândia, UFU, Uberlândia, MG, Brazil.
authors: Daiana Silva
authors: DS
authors: Lopes
authors: Instituto de Genética e Bioquímica, Universidade Federal de Uberlândia, UFU, Uberlândia, MG, Brazil; INCT, Instituto Nacional de Ciência e Tecnologia em Nano-Biofarmacêutica, Brazil. Electronic address: veridiana@ingeb.ufu.br.
authors: Veridiana M
authors: VM
authors: Rodrigues
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D017837:Bothrops; D003435:Crotalid Venoms / Q000737:chemistry; D017839:Crotalus / Q000097:blood*; D006023:Glycoproteins / Q000737:chemistry* / Q000302:isolation & purification; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D064801:Phospholipase A2 Inhibitors / Q000737:chemistry* / Q000302:isolation & purification; D054467:Phospholipases A2 / Q000302:isolation & purification; D030162:Reptilian Proteins / Q000737:chemistry* / Q000302:isolation & purification; D016415:Sequence Alignment; D020539:Sequence Analysis, Protein; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D003435:Crotalid Venoms; D006023:Glycoproteins; D064801:Phospholipase A2 Inhibitors; D030162:Reptilian Proteins; C087438:phospholipase A2 inhibitor protein, Crotalus; D054467:Phospholipases A2
keywords: Crotalus durissus collilineatus; Inhibitor γ-type; Phospholipase A(2); Phospholipase A(2) inhibitors; Snake