Ponce-Soto Luis Alberto, Martins-de-Souza Daniel, Novello José Camillo, Marangoni Sergio
Departamento de Bioquímica, Instituto de Biologia (IB), Universidade Estadual de Campinas (UNICAMP), CP 6109, CEP 13083-970, Campinas, São Paulo, Brasil.
Protein J. 2007 Dec;26(8):533-40. doi: 10.1007/s10930-007-9094-z.
In this work, we isolated the two new crotamine isoforms from the Crotalus durissus cumanensis rattlesnake venom and its "in vitro" neurotoxic, myotoxic and lethality (DL(50)) intracerebroventricular (i.c.v.) effects were characterized. These proteins were named IV-2 and IV-3 and were purified by combination of two chromatographic steps on molecular exclusion chromatography on Superdex 75 and reverse phase HPLC (mu-Bondapack C18). The molecular mass of the crotamine isoforms was 4905.96 Da for isoform IV-2 and 4956.97 Da for IV-3 and, as determined by mass spectrometry, and both contained six Cys residues. Enzymatic hydrolysis followed by de novo sequencing by tandem mass spectrometry was used to determine the primary structure of both isoforms. The positions of five sequenced tryptic peptides, including the N-terminal of the isoform IV-2 and four from isoform IV-3 were deduced by comparison with a homologous protein from the crotamine family. The isoforms IV-2 and IV-3 had a sequence of amino acids of 42 amino acid residues IV-2: YKRCHIKGGH CFPKEKLICI PPSSDIGKMD CPWKRKCCKK RS and pI value 9.54 and IV-3: YKQCHKKGGH CFPKEVLICI PPSSDFGKMD CRWKRKCCKK RS with a pI value of 9.54. This protein showed high molecular amino acid sequence identity with other crotamine-like proteins from Crotalus durissus terrificus. These new crotamine isoforms induced potent blockade of neuromuscular transmission in young chicken biventer cervicis preparation and potent myotoxic effect. In mice, both isoforms induced myonecrosis, upon intramuscular or subcutaneous injections. These activities were modulated by the presence of positively charged amino acid residues. The LD(50) of isoform IV-2 was 0.07 mg/kg and isoform IV-3 was 0.06 mg/kg the animal weight, by i.c.v. route.
在本研究中，我们从杜氏猪鼻蝰蛇毒中分离出两种新的响尾蛇胺同工型，并对其“体外”神经毒性、肌毒性及脑室内注射（i.c.v.）致死率（LD(50)）效应进行了表征。这些蛋白质被命名为IV-2和IV-3，并通过在Superdex 75上的分子排阻色谱和反相高效液相色谱（μ-Bondapack C18）两步色谱法相结合进行纯化。通过质谱测定，响尾蛇胺同工型IV-2的分子量为4905.96 Da，IV-3为4956.97 Da，且二者均含有六个半胱氨酸残基。采用酶解后串联质谱从头测序的方法确定了两种同工型的一级结构。通过与响尾蛇胺家族的同源蛋白比较，推导了五个测序胰蛋白酶肽段的位置，包括同工型IV-2的N端和来自同工型IV-3的四个肽段。同工型IV-2和IV-3具有42个氨基酸残基的氨基酸序列：IV-2：YKRCHIKGGH CFPKEKLICI PPSSDIGKMD CPWKRKCCKK RS，pI值为9.54；IV-3：YKQCHKKGGH CFPKEVLICI PPSSDFGKMD CRWKRKCCKK RS，pI值为9.54。该蛋白质与杜氏猪鼻蝰的其他类响尾蛇胺蛋白具有高度的分子氨基酸序列同一性。这些新的响尾蛇胺同工型在幼鸡二腹肌制备中诱导了强大的神经肌肉传递阻滞和强大的肌毒性作用。在小鼠中，两种同工型经肌肉或皮下注射后均诱导了肌坏死。这些活性受到带正电荷氨基酸残基的影响。通过脑室内途径给药时，同工型IV-2的LD(50)为0.07 mg/kg动物体重，同工型IV-3为0.06 mg/kg动物体重。
