RESUMO
A combination of hydrophobic chromatography on phenyl-Sepharose and reversed phase HPLC was used to purify individual tRNAs with high specific activity. The efficiency of chromatographic separation was enhanced by biochemical manipulations of the tRNA molecule, such as aminoacylation, formylation of the aminoacyl moiety and enzymatic deacylation. Optimal combinations are presented for three different cases. (i) tRNA(Phe) from Escherichia coli. This species was isolated by a combination of low pressure phenyl-Sepharose hydrophobic chromatography with RP-HPLC. (ii) tRNA(Ile) from E. coli: Aminoacylation increases the retention time for this tRNA in RP-HPLC. The recovered acylated intermediate is deacylated by reversion of the aminoacylation reaction and submitted to a second RP-HPLC run, in which deacylated tRNA(Ile) is recovered with high specific activity. (iii) tRNA(i)(Met) from Saccharomyces cerevisiae. The aminoacylated form of this tRNA is unstable. To increase stability, the aminoacylated form was formylated using E.coli: enzymes and, after one RP-HPLC step, the formylated derivative was deacylated using peptidyl-tRNA hydrolase from E.COLI: The tRNA(i)(Met) recovered after a second RP-HPLC run exhibited electrophoretic homogeneity and high specific activity upon aminoacylation. These combinations of chromatographic separation and biochemical modification can be readily adapted to the large-scale isolation of any particular tRNA.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Cromatografia/métodos , RNA de Transferência/isolamento & purificação , Acilação , Hidrolases de Éster Carboxílico/isolamento & purificação , Hidrolases de Éster Carboxílico/metabolismo , Cromatografia em Agarose , Escherichia coli/genética , RNA Bacteriano/isolamento & purificação , RNA Fúngico/isolamento & purificação , RNA de Transferência/química , RNA de Transferência de Isoleucina/isolamento & purificação , RNA de Transferência de Metionina/isolamento & purificação , RNA de Transferência de Fenilalanina/isolamento & purificação , Saccharomyces cerevisiae/genética , Sensibilidade e Especificidade , Fatores de TempoRESUMO
BACKGROUND: Only a few studies have investigated the clinical role of food allergens, especially the relationship between sensitization to a given allergen and occurrence of adverse reactions when eating the relevant food item. OBJECTIVE: This study evaluated the clinical role of the allergens of Brazil nut by comparing the patterns of IgE binding in sera from 11 patients with anaphylaxis after eating Brazil nuts with those from 10 subjects with no symptoms to this food item. Both groups had specific IgE to Brazil nut. METHODS: Allergens in the in-house extract of Brazil nut were identified by SDS-PAGE/immunoblotting, the major allergen was purified by HPLC, and its N-terminal sequence was determined by a protein sequencer. RESULTS: SDS-PAGE/immunoblotting detected a number of allergenic components with molecular weights ranging from 4 to 58 kd. All sera from symptomatic patients recognized a 9-kd allergen corresponding (as established by amino acid sequencing) to a 2S albumin already described as a major allergen of Brazil nut, whereas the other allergens each bound IgE from less than 50% of sera. No sera from asymptomatic subjects showed IgE binding to the 9-kd allergen, but they did recognize components from 25 to 58 kd, which are minor allergens. CONCLUSIONS: These findings indicate that the allergen underlying clinical reactions to Brazil nut is a 2S albumin of 9 kd and that in vitro reactivity to this allergen identifies subjects who react in vivo to ingestion of this food.