Latex of plumeria rubra L. (Jasmim): protein Profile, enzymatic characterization and action against insects.

ARAÚJO, E. S. Látex de Plumeria rubra L. (jasmim): perfil protéico, caracterização enzimática e ação contra insetos. 2009. 90 f. Dissertação (Mestrado em Bioquímica) - Centro de Ciências, Universidade Federal do Ceará, Fortaleza, 2009.Plumeria rubra L. is a laticífera plant belonging to the family Apocynaceae popularly known as Jasmim. For spicy and fragrant flowers make this tree is commonly seen ornate squares and gardens of residential urban centers. Laticífer plants are well known for producing a fluid endogenously generally milky in appearance - latex - that exudates from the plant when it undergoes some type of injury, either by mechanical damage or attack by predators. Latex is a material that has been the subject of biochemical and pharmacological studies show that it is a promising source of compounds with potential biotechnological application. In this study the latex of P. rubra was the subject of biochemical and biological research with emphasis on the search for proteins that could make any deleterious action against agricultural insect pests. Laboratory procedures that employ centrifugation and dialysis allowed the fractionation of latex into three distinct components: rubber (BL), proteins (PLPr) and low molecular weight molecules (DL). The PLPr fraction was subject to biochemical and enzymatic characterization and was used in bioassays with the bean weevil-of-rope, Callosobruchus maculatus and the fruit fly, Ceratitis capitata. The full latex and its fractions were used in testing the repellency of oviposition of C. maculatus and Zabrotes subfasciatus. The BL fraction is the major component of latex, 70 %, while PLPr and DL fractions are about 15 % each one. The content of soluble protein in PLPr fraction was 0.33 mg / mL. Espectrometric and electrophoretic analysis revealed the presence of proteins with molecular weights ranging from 12 to 117 kDa, with a maximum of protein with 26 kDa and pI <6.0. The characterization showed that the enzyme antioxidativas enzymes superoxide dismutase and peroxidase were detected in the protein fraction, as well as activity chitinases. Proteins of latex were able to degrade azocasein (nonspecific substrate) and BANA (specific substrate for cysteine proteases). The proteolytic enzymes were detected mainly of type cysteine and to a lesser extent, serine proteases. The pH and temperature optimum for this activity was 6.0 and 37 ºC respectively, where higher values of temperature cancel the activity. Through experiments with artificial diet can be observed that the protein fraction of latex in 0.4 % was able to reduce by 50 % the survival and, in 0.1 %, reduce the by 50 % the weight gain of larvae of C. maculatus. When these proteins were denatured by heating the larval development was similar to the control, suggesting that the maintenance of protein structure is essential for the observed effect. Proteins of latex were not digested by endogenous proteases of the digestive tract of larvae, suggesting that they would be free to cause deleterious effects. No effect on the development of the larvae of C. capitata was observed when the proteins were added to the diet, even at a concentration of 4 %. Whole latex when adsorbed in bean seeds, showed repellent activity on oviposition of both bruchid, being more evident in Z. subfasciatus. The repellent activity was observed time and dose dependent. The latex was unable to affect the viability of eggs or larval development. The PLPr and DL fractions showed no repellent activity, showing that neither protein nor molecules with low molecular weight are involved in this action. The insects, when exposed directly to the latex for a long period of time had not affected their fecundity or oviposition.Plumeria rubra L. é uma planta laticífera pertencente à família Apocynaceae popularmente conhecida como Jasmim. Por apresentar flores vistosas e perfumadas, essa árvore é comumente vista ornamentando praças e jardins residenciais dos grandes centros urbanos. Plantas laticíferas são assim denominadas por que produzem endogenamente um fluido de aspecto geralmente leitoso - o látex - que é exsudado da planta quando esta sofre algum tipo de ferimento, seja por dano mecânico ou por ataque de predadores. O látex é um material vegetal que tem sido alvo de estudos bioquímicos e farmacológicos que mostram ser ele uma fonte promissora de compostos com potencial aplicação biotecnológica. No presente trabalho o látex de P. rubra foi alvo de investigações bioquímicas e biológicas com ênfase na pesquisa de proteínas que pudessem apresentar alguma ação deletéria contra insetos pragas agrícolas. Procedimentos laboratoriais que empregam centrifugações e diálises permitiram o fracionamento do látex em três componentes distintos: borracha (BL), proteínas (PLPr) e moléculas de baixa massa molecular (DL). A fração PLPr foi alvo de caracterização bioquímica e enzimática e foi utilizada em bioensaios com o caruncho do feijão-de-corda, Callosobruchus maculatus e com a mosca-das-frutas, Ceratitis capitata. O látex íntegro e suas frações foram utilizados em ensaios de repelência da ovoposição de C. maculatus e Zabrotes subfasciatus. A fração BL é o componente majoritário do látex, 70 %, enquanto as frações PLPr e DL constituem cerca de 15 % cada uma. O teor de proteínas solúveis na fração PLPr foi de 0,33 mg/mL. Análises eletroforética e espectrométricas revelaram a presença de proteínas com massas moleculares que variaram de 12 a 117 kDa, com um máximo de proteínas com 26 kDa e pI