RESUMO
Hemoglobin I (HbI) from Lucina pectinata is a monomeric protein composed of 143 amino acids with high sulfide affinity. Its unique heme pocket contains three residues not commonly found in vertebrate globins: Phe 29 (B10), Gln 64 (E7), and Phe 68 (E11), which are thought to be important for high affinity for hydrogen sulfide. Recombinant HbI (rHbI) and several site-directed mutants were cloned and expressed in Escherichia coli yielding high amounts of protein. The highest rHbI protein yield was obtained when the HbI cDNA was cloned into the pET28 (a+) expression vector, transformed into BLi5 cells, the induction performed with 1 mM IPTG at 30 degrees C and TB medium was supplemented with 30 microg/mL hemin chloride and 1% glucose. The highest yield obtained of HbI was 32 mg/L of culture using Fernbach flasks. UV/Visible spectral analysis showed that rHbI binds heme and ESI-MS shows that its molecular weight corresponds to the expected size. Kinetic studies with H2S confirmed that rHbI and HbI have identical binding properties, where the kON for the clam's Hb is 2.73x10(4)M-1s-1 and for rHbI is 2.43x10(4)M-1s-1.
Assuntos
Escherichia coli/genética , Hemoglobinas , Sulfeto de Hidrogênio/química , Sequência de Aminoácidos , Animais , Bivalves/metabolismo , Clonagem Molecular , Regulação Bacteriana da Expressão Gênica , Vetores Genéticos/genética , Hemoglobinas/biossíntese , Hemoglobinas/genética , Hemoglobinas/isolamento & purificação , Cinética , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida/genética , Ligação Proteica , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Espectrometria de Massas por Ionização por Electrospray , Espectrofotometria Ultravioleta , TemperaturaRESUMO
The clam Lucina pectinata inhabits the sulfide-rich west coast of the island of Puerto Rico. It contains three different hemoglobins. Hemoglobin I (HbI), which is monomeric at all concentrations, carries H2S in its ferric state. Overexpression of recombinant HbI from Lucina pectinata in BL21STAR(DE3) Escherichia coli cells was performed in the presence of delta-aminolevulinic acid (delta-ALA). Purification of the protein was achieved using FPLC anion exchange and size exclusion chromatography. Functional characterization of the recombinant holo-protein was assessed by detection of the protein heme O2, CO, and H2S derivatives by UV-Vis spectroscopy, with Soret maxima at 416, 421, and 425 nm, respectively. The results indicated that the recombinant HbI binds H2S and forms a heme sulfide complex like the HbI wild-type hemoglobin. Kinetic measurements were performed to determine the H2S affinity of the recombinant HbI. The H2S dissociation and association rate constants were 0.055 x 10(-3)s(-1) and 0.068 x 10(5) M(-1)s(-1), respectively. The H2S affinity constant of the recombinant HbI (0.124 x 10(9) M(-1)) is eightfold lower than that of the native clam HbI reported earlier. This effect is attributed mostly to the first of two missense mutations [Met 61 (E4)-->Val 61 and Ile101 (FG4)-->Val 101] and additional amino acids present in our construct as demonstrated by measurements of the association rate with a new construct lacking most of the additional residues and the missense mutations. The elimination of these residues restores the similarity between the expressed and wild-type hemoglobins, as evidenced by H2S association kinetics. A pH dependence on the H2S association rate was also contributing to the overall affinity constant and was taken into account in the measurements of the functional properties of the new HbI construct.