Caracterização da interação de inositol hexafosfato e cloreto com hemoglobina humana: aspectos energéticos e estruturais
Carregando...
Data
2010-01-22
Autores
Silva, Vanessa de Cássia Teixeira da [UNESP]
Título da Revista
ISSN da Revista
Título de Volume
Editor
Universidade Estadual Paulista (Unesp)
Resumo
A ligação do O2 à Hemoglobina (Hb) é um processo regulado por interações alostéricas. Os íons Inositol hexafosfato (IHP) e cloreto, e prótons, são efetores alostéricos que estabilizam a Hb na estrutura quaternária T, diminuindo a afinidade da proteina pelo O2 em meio alcalino. A ligação de IHP também altera a cooperatividade de algumas hemoglobinas como, por exemplo a hemoglobina desArg, uma variante da HbAo da qual o resíduo arginina 141 das cadeia alfas foram retirados enzimaticamente. Estas argininas são prováveis sitios de interação com ions cloreto. Neste trabalho investigou-se a influência que a ligação de ânions Cl - e/ou IHP à Hb nas propriedades funcionais e conformacionais da proteína. Foi determinado o efeito destas interações na afinidade e cooperatividade de ligação de oxigênio à proteína, na liberação de prótons com a oxigenação ( efeito Bohr ), e na conformação da proteína. O papel de resíduos específicos nestes efeitos foi investigado comparando resultados obtidos com HbAo, Hb-desArg, e Hb Chesapeake (R92L), um mutante natural na qual a arginina 92 esta substituída por leucina. Os resultados mostraram que a retirada do resíduo C-terminal da cadeia alfa, arginina 141, suprimiu a cooperatividade da hemoglobina, diminui o número de prótons liberados durante a oxigenação, e que esta liberação de prótons foi a mesma na presença ou ausência de íons cloreto. Ainda, a retirada deste resíduo não interferiu no efeito Bohr dependente de IHP. Os resultados mostraram que a saturação de oxigênio ligados à HbA0 na presença de IHP não induziu a mudança na estrutura quaternária observada na ausência deste ânion, e na sua presença em meio com NaCl. Apesar disto, a cooperatividade da ligação é mantida. A arg-141α tem papel fundamental na estabilização pelo IHP da estrutura quaternária...
The hemoglobin (Hb) oxygenation is a molecular process regulated by allosteric interactions. Inositol hexaphosphate (IHP), chloride and protons are heterotropic effectors that stabilizes the deoxy state, the quaternary structure T, decreasing O2 affinity in alkaline solution. IHP binding also modifies cooperativity of several Hbs, such as desArg Hemoglobin, a variant form of HbA which is obtained by enzymatic cleavage of the two Arg-141alfa residues. This residue has been identified to be a Cl - binding site on Hb. This work follows up previous studies of the structure- function regulation of Hb oxygenation by anions Cl - and/or IHP. We measured the effect of these interactions on cooperativity and oxygen affinity, protons release upon oxygenation (Bohr Effect), and protein conformation. The role of specific residues on this effect was investigated trough results obtained with HbA, desArg Hemglobin and Hemoglobin Chesapeake (R92L), a natural mutant in which Arg- 92α is changed to Leu. The results showed that the suppression of C-terminal Arg-141 implies in non- cooperative O2-binding to Hemoglobin, lowering the protons released during oxygenation, and the protons release remains the same on chloride absence and presence. The removal of this residue didn´t affect the Bohr Effect on IHP presence. The results showed that O2 binding in the presence of IHP don´t induce quaternary structure transition observed in the absence of anions. Nevetheless, the cooperativity was maintained. The arg-141 have a main role on IHP stabilization of oxy and deoxy protein quaternary structure T. This conclusion is supported by results desArg Hemoglobin oxygenation determined in IHP presence, with and without NaCl. Calorimeters assays of IHP interaction with three Hemoglobin species, carried through Isothermal Titration Calorimetry confirm the Arg-141 importance on oxygenated... (Complete abstract click electronic access below)
The hemoglobin (Hb) oxygenation is a molecular process regulated by allosteric interactions. Inositol hexaphosphate (IHP), chloride and protons are heterotropic effectors that stabilizes the deoxy state, the quaternary structure T, decreasing O2 affinity in alkaline solution. IHP binding also modifies cooperativity of several Hbs, such as desArg Hemoglobin, a variant form of HbA which is obtained by enzymatic cleavage of the two Arg-141alfa residues. This residue has been identified to be a Cl - binding site on Hb. This work follows up previous studies of the structure- function regulation of Hb oxygenation by anions Cl - and/or IHP. We measured the effect of these interactions on cooperativity and oxygen affinity, protons release upon oxygenation (Bohr Effect), and protein conformation. The role of specific residues on this effect was investigated trough results obtained with HbA, desArg Hemglobin and Hemoglobin Chesapeake (R92L), a natural mutant in which Arg- 92α is changed to Leu. The results showed that the suppression of C-terminal Arg-141 implies in non- cooperative O2-binding to Hemoglobin, lowering the protons released during oxygenation, and the protons release remains the same on chloride absence and presence. The removal of this residue didn´t affect the Bohr Effect on IHP presence. The results showed that O2 binding in the presence of IHP don´t induce quaternary structure transition observed in the absence of anions. Nevetheless, the cooperativity was maintained. The arg-141 have a main role on IHP stabilization of oxy and deoxy protein quaternary structure T. This conclusion is supported by results desArg Hemoglobin oxygenation determined in IHP presence, with and without NaCl. Calorimeters assays of IHP interaction with three Hemoglobin species, carried through Isothermal Titration Calorimetry confirm the Arg-141 importance on oxygenated... (Complete abstract click electronic access below)
Descrição
Palavras-chave
Biofísica, Biologia molecular, Hemoglobina, Anions, Biofísica molecular, Hemoglobin, Anions
Como citar
SILVA, Vanessa de Cássia Teixeira da. Caracterização da interação de inositol hexafosfato e cloreto com hemoglobina humana: aspectos energéticos e estruturais. 2010. 81 f. Dissertação (mestrado) - Universidade Estadual Paulista, Instituto de Biociências, Letras e Ciências Exatas, 2010.