Resumo
Este trabalho apresenta os resultados de proteínas associadas ao mercúrio em amostras de tecido muscular e hepático de pirarucu (Arapaima gigas) e piranha preta (Serrasalmus rhombeus) oriundos do complexo hidrelétrico de Jirau, na Bacia do Rio Madeira, região amazônica do Brasil. O proteôma do músculo e fígado dessas espécies foi obtido por eletroforese bidimensional em gel de poliacrilamida (2D PAGE). O mercúrio presente nos spots proteicos foi determinado por espectrometria de absorção atômica em forno de grafite (GFAAS) após mineralização ácida assistida por banho de ultrassom. Os spots proteicos que apresentaram mercúrio foram caracterizados por espectrometria de massas por ionização com eletrospray em sequência (ESI- MS/MS) após digestão tríptica. As determinações GFAAS indicaram que a maior parte do mercúrio está ligada a fração proteica com massa molar (Mm) inferior a 90 kDa. As concentrações de mercúrio nos spots apresentaram-se na faixa de 4,07 164,63 µg g-1 no tecido muscular de pirarucu; 0,86 25,34 µg g-1 no tecido hepático de pirarucu; 7,67 156,18 µg g-1 no tecido muscular de piranha preta e 2,17 31,42 µg g-1 no tecido hepático de piranha preta. A análise por ESI-MS/MS permitiu caracterizar em dezenove spots proteicos as seguintes proteínas e/ou enzimas: Triosephosphate isomerase, Fructosebisphosphate aldolase, Ckmb protein,, Cofilin 2 (Muscle), Actin_ alpha_ cardiac muscle 1a, Actin_ alpha 1_ skeletal muscle, Novel protein similar to zebrafish hemoglobin alpha adult 1 (Hbaa1), Hemoglobin subunit alpha, Embryonic 1 beta-globin, Embryonic globin beta e2, Hydroxyacyl-Coenzyme A dehydrogenase, Adenosylhomocysteinase, Glycine N-methyltransferase, Peroxiredoxin 2, Enolase 1_ (Alpha), Fatty acid binding protein 11a, Parvalbumin 3, 3-hydroxyanthranilate 3_4-dioxygenase e Glutathione peroxidase.
This paper presents a simple, fast and sensitive method to determine mercury in muscle and liver tissue samples of fish from the Brazilian Amazon by GFAAS through the direct introduction of sample slurries into the spectrometers graphite tube. The samples of muscle and/or liver tissues were lyophilized and macerated using a mortar and pestle in the presence of liquid nitrogen. After this treatment, it was possible to obtain particles with a diameter of 60 µm. The slurry samples were prepared by mixing 20 mg of tissue (muscle end/or liver) with 1 mL of a solution containing 0.05 % v/v Triton X-100, 0.50 % v/v suprapur HNO3 and 100 mg L-1 zirconium directly in the spectrometers automatic sampling glass. Mercury standard solutions with concentrations ranging between 0.25 and 2.00 g L1 were prepared under the same conditions as the slurry samples and used to generate the analytical curve. After sonicating the slurry samples and/or mercury standard solutions for 20 seconds, 20 L was injected into the graphite tube, whose internal wall was lined with a tungsten carbide film that acted as a permanent chemical modifier. Under these conditions, it was possible to obtain thermal stabilization of the mercury up to an atomization temperature of 1700 °C in the GFAAS assay. The proposed method was validated by mercury determination in slurries of fish protein certified reference material DORM-4 and liver certified reference material DOLT-4. The calculated limits of detection (LOD) and quantification (LOQ) in relation to DORM-4 and DOLT-4 were on the order of 0.014 - 0016 mg kg-1 and 0.045 0.0051 g kg-1, respectively.
Resumo
Este trabalho apresenta os resultados de fracionamento de proteínas ligadas ao mercúrio em amostras de músculo de tucunaré (Cichla spp.) e filhote (Brachyplatystoma filamentosum) oriundos do complexo hidrelétrico de Jirau, na Bacia do Rio Madeira, região amazônica do Brasil. O proteoma do músculo dessas espécies foi separado por eletroforese bidimensional em gel de poliacrilamida (PAGE 2D). O mercúrio presente nos spots proteicos foi determinado por espectrometria de absorção atômica em forno de grafite (GFAAS) após mineralização ácida assistida por banho de ultra-som. Os spots proteicos que apresentaram mercúrio foram caracterizados por espectrometria de massas por ionização com eletrospray em sequência (ESI- MS/MS) após digestão tríptica. As determinações GFAAS indicaram que 65% do mercúrio está ligada à fração proteica com massa molar (Mm) inferior a 90 kDa. As concentrações de mercúrio nos spots apresentaram-se na faixa de 13,60 ¿ 17,10 mg g -1 . Com base nas concentrações de mercúrio, foi possível estimar que os spots proteicos continham aproximadamente um átomo de mercúrio por molécula de proteína. A análise por ESI-MS / MS permitiu caracterizar em dez spots proteicos as seguintes proteínas e/ou enzimas: parvalbumina-2 (Mm = 12,40, pI = 3,80); parvalbumina alfa (Mm = 12,40, pI = 3,80); parvalbumina beta (Mm = 12,40, pI = 3,80); ubiquitina-40S proteína ribossômica S27a (Mm = 11,60, pI = 6,10); GTP cyclohydrolase 1 proteína reguladora de feedback (Mm = 13,00, pI = 4,00) e proteína de transmembrana 186 (Mm = 14,00, pI = 4,70).
This paper presents the results of mercury fractionation in muscle samples of tucunaré (Cichla spp.) and filhote (Brachyplatystoma filamentosum) from the JIRAU Hydroelectric Power Plant in the Madeira River Basin in the Amazon region of Brazil. The proteome of the fishes muscle was separated by two-dimensional polyacrylamide gel electrophoresis (2D PAGE). The mercury present in the protein spots was determined by graphite furnace atomic absorption spectrometry (GFAAS) after acid mineralization in an ultrasound bath. The protein spots in which the presence of mercury was detected were characterised by electrospray ionisation tandem mass spectrometry (ESI-MS/MS) after tryptic digestion. The GFAAS determinations indicated that 65% of the mercury was linked to the protein fraction with a molar mass (Mm) of less than 90 kDa. The mercury concentration in the eleven spots in this protein fraction was present were in the range 13.60 to 17.10 mg g -1 . Based on mercury concentrations, it was possible to estimate that the protein spots contained approximately one atom of mercury per protein molecule. Analysis by ESI-MS / MS protein allowed the characterization of ten spots as proteins and / or following enzymes: parvalbumin-2 (MW = 12.40, pI = 3.80); parvalbumin alpha (Mm = 12.40, pI = 3.80); parvalbumin beta (Mm = 12.40, pI = 3.80); ubiquitin-40S ribosomal protein S27a (Mm = 11.60, pI = 6.10); GTP cyclohydrolase 1 regulatory protein of feedback (Mm = 13.00, pI = 4.00) and transmembrane protein 186 (Mm = 14.00, pI = 4.70).