Molecular Cloning and Characterization of a (Lys)6-Tagged Sulfide-Reactive Hemoglobin I from Lucina pectinata.
A poly-Lys tag was fused to the Lucina pectinata hemoglobin I (HbI) coding sequence and purified using an efficient and fast process. HbI is a hemeprotein that binds hydrogen sulfide (H2S) with high affinity and it has been used to understand physiologically relevant reactions of this signaling molecule. The (Lys)6-tagged rHbI construct was expressed in E. coli and purified by immobilization on a cation exchange matrix, followed by size-exclusion chromatography. The identity, structure, and function of the (Lys)6-tagged rHbI were assessed by mass spectrometry, small and wide X-ray scattering, optical spectroscopy, and kinetic analysis. The scattering and spectroscopic results showed that the (Lys)6-tagged rHbI is structurally and functionally analogous to the native protein as well as to the (His)6-tagged rHbI. Kinetics studies with H2S indicated that the association (k on) and dissociation (k off) rate constants were 1.4 × 10(5)/M/s and 0.1 × 10(-3)/s, respectively. This results confirmed that the (Lys)6-tagged rHbI binds H2S with the same high affinity as its homologue.
Ramonita
R
Díaz-Ayala
Andrés
A
Moya-Rodríguez
Ruth
R
Pietri
Carmen L
CL
Cadilla
Juan
J
López-Garriga
D000595:Amino Acid Sequence; D000818:Animals; D049872:Bivalvia / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D005786:Gene Expression Regulation; D006420:Hemeproteins / Q000737:chemistry; D006454:Hemoglobins / Q000235:genetics / Q000378:metabolism*; D006862:Hydrogen Sulfide / Q000737:chemistry; D050576:Membrane Fusion Proteins / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D011487:Protein Conformation; D012542:Scattering, Radiation; D013440:Sulfides / Q000737:chemistry*
D006420:Hemeproteins; D006454:Hemoglobins; D050576:Membrane Fusion Proteins; D013440:Sulfides; C026236:hemoglobin I, non-mammalian; D006862:Hydrogen Sulfide