Caracterização e imobilização da hemoglobina extracelular de Amynthas gracilis em substrato sólido visando a elaboração de biossensores de contaminação ambiental
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Data
2018-03-09
Autores
Souza, Claudemir Oliveira
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Editor
Universidade Estadual Paulista (Unesp)
Resumo
As hemoproteínas desempenham um papel vital nos organismos. Dentre elas, as hemoglobinas extracelulares gigantes (Hbs) se destacam por sua alta cooperatividade, alta estabilidade oligomérica, grande afinidade para ligação com oxigênio e resistência à oxidação. Devido estas propriedades, as Hbs apresentam um grande potencial biotecnológico, como é o caso de estudos buscando protótipos de substituição sanguínea e elaboração de biossensores. Porém, o uso das hemoglobinas gigantes em biossensores para detecção de metais pesados ainda não é encontrado na literatura, este tipo de biossensor agiliza e diminui o custo das análises de empresas e agências de fiscalização ambiental, obtendo o resultado in loco. Diante do exposto, o objetivo deste trabalho foi verificar a sensibilidade da hemoglobina de Amynthas gracilis (HbAg) com os metais cobre e cádmio em diferentes valores de pH e imobilizá-la em substrato sólido com o auxílio dos seguintes polímeros: o catiônico polietilenoimina (PEI) e o aniônico hidrocloreto de polialilamina (PAH). Os estudos por absorção ótica, espalhamento de luz (LSI), dicroísmo circular (CD) e fluorescência da interação da HbAg em função da concentração do metal pesado cobre no pH 7,0 mostraram que a concentração de 30 μmol.L-1 é uma concentração crítica onde observa-se uma maior oxidação do grupo heme, agregação e dissociação da proteína, além de alteração em suas α- hélices. No pH 5,0 foi observada oxidação do grupo heme mas sem alterações nas estruturas secundárias da HbAg e formação de agregados. As análises dos espectros de absorção ótica, CD, fluorescência e LSI da HbAg na presença de diferentes concentrações do metal pesado cadmio não apresentaram mudanças significativas quando comparadas com o espectro da HbAg nativa. A imobilização da HbAg em PEI no pH 7,0 foi monitorada por absorção ótica e voltametria cíclica, entretanto a HbAg não foi possível imobilizar a HbAg no polímero PAH no mesmo valor de pH, sugerindo que a interação da HbAg com os biopolímeros se deve por interações eletrostáticas. A imobilização da HbAg em PEI manteve as características da hemoglobina em pH 7,0. A hemoglobina apresentou respostas ao cobre nas medidas de voltametria cíclica realizadas em concentrações de até 20 μmol.L-1 do metal, evidenciando o grande potencial de biossensoriamento do sensor fabricado. Assim, nestes estudos foi possível verificar que a HbAg na presença de cobre em pH 7,0 se oxida, agrega e dissocia, enquanto que em pH 5,0 ela apresenta apenas oxidação. Na presença de cádmio, a HbAg não apresentou nenhuma alteração através das técnicas avaliadas. Com isso, sugere-se que a HbAg pode ser utilizada como um biossensor de indicação de presença de concentrações micromolares do metal pesado cobre.
Hemoproteins are a group of proteins that play a vital role in organisms. Among them, giant extracellular hemoglobins (Hbs) stand out for their high cooperativity, high oligomeric stability, high affinity for oxygen binding and resistance to oxidation. Due to these properties, the Hbs show a great biotechnological potential, as is the case of studies searching for prototypes of blood replacement and preparation of biosensors. However, the use of giant hemoglobins in biosensors for heavy metals detection is not yet reported on the literature, this kind of biosensors speeds up and lowers costs of analysis of companies and environmental inspection agencies, obtaining on the spot result. Thus, the aim of this work was verified the sensibility of HbAg with the metals copper and cadmium on different pH values and it's immobilization in solid surfaces with the polymers: the cationic polietilenimine (PEI) and anionic polyallylamine hydrochloride (PAH). Studies of optical absorption (UVVis), light scattering (LSI), circular dichroism (CD) and fluorescence of HbAg interaction with copper at pH 7.0 shown that 30 μmol.L-1 it’s a critical concentration where observed a great oxidation of group heme, aggregation and protein dissociation as well as alteration on your α-hélices. In pH 5.0 was observed oxidation of heme group but without alterations in HbAg secondary structure and no aggregates formation. UV-Vis spectra analysis, CD, fluorescence and LSI of HbAg at different concentrations of cadmium doesn’t shown significant changes on native HbAg spectra. The HbAg immobilization on PEI pH 7,0 has monitored by optical absorption and cyclic voltammetry, however it was not possible to immobilize the HbAg in the PAH polymer at the same pH value, suggesting that HbAg interaction with biopolymers is due to electrostatic ligations. The HbAg Immobilization in PEI maintained the hemoglobin characteristics at pH 7.0. The Hemoglobin shown responses to copper at concentrations of up to 20 μmol.L-1 of the metal in cyclic voltammetry measurements performed, demonstrated the great potential of the fabricated biosensor. Thus, in these studies it was possible to verify that HbAg on copper presence at pH 7.0 oxidizes, to aggregates and dissociates whereas at pH 5.0 it presents only oxidation. In the presence of cadmium, the HbAg showed no change through the techniques evaluated. Thereby, it is suggested that HbAg can be used as a biosensor indicating the presence of micromolar concentrations of copper heavy metal.
Hemoproteins are a group of proteins that play a vital role in organisms. Among them, giant extracellular hemoglobins (Hbs) stand out for their high cooperativity, high oligomeric stability, high affinity for oxygen binding and resistance to oxidation. Due to these properties, the Hbs show a great biotechnological potential, as is the case of studies searching for prototypes of blood replacement and preparation of biosensors. However, the use of giant hemoglobins in biosensors for heavy metals detection is not yet reported on the literature, this kind of biosensors speeds up and lowers costs of analysis of companies and environmental inspection agencies, obtaining on the spot result. Thus, the aim of this work was verified the sensibility of HbAg with the metals copper and cadmium on different pH values and it's immobilization in solid surfaces with the polymers: the cationic polietilenimine (PEI) and anionic polyallylamine hydrochloride (PAH). Studies of optical absorption (UVVis), light scattering (LSI), circular dichroism (CD) and fluorescence of HbAg interaction with copper at pH 7.0 shown that 30 μmol.L-1 it’s a critical concentration where observed a great oxidation of group heme, aggregation and protein dissociation as well as alteration on your α-hélices. In pH 5.0 was observed oxidation of heme group but without alterations in HbAg secondary structure and no aggregates formation. UV-Vis spectra analysis, CD, fluorescence and LSI of HbAg at different concentrations of cadmium doesn’t shown significant changes on native HbAg spectra. The HbAg immobilization on PEI pH 7,0 has monitored by optical absorption and cyclic voltammetry, however it was not possible to immobilize the HbAg in the PAH polymer at the same pH value, suggesting that HbAg interaction with biopolymers is due to electrostatic ligations. The HbAg Immobilization in PEI maintained the hemoglobin characteristics at pH 7.0. The Hemoglobin shown responses to copper at concentrations of up to 20 μmol.L-1 of the metal in cyclic voltammetry measurements performed, demonstrated the great potential of the fabricated biosensor. Thus, in these studies it was possible to verify that HbAg on copper presence at pH 7.0 oxidizes, to aggregates and dissociates whereas at pH 5.0 it presents only oxidation. In the presence of cadmium, the HbAg showed no change through the techniques evaluated. Thereby, it is suggested that HbAg can be used as a biosensor indicating the presence of micromolar concentrations of copper heavy metal.
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Palavras-chave
Hemoglobina, Biossensores, Biopolímeros, Solos - Poluição, Hemoglobin, Biosensors, Heavy metals, Metais pesados