| Lucina pectinata is a bivalve mollusk that inhabits sulfide rich sediments along, the west coast of Puerto Rico. This clam contains three hemoglobins that deliver and transport oxygen (HbII and III) and hydrogen sulfide (HbI) to chemoautotrophic bacteria. The HbII is an oxygen reactive protein in which the oxygen is anchored to the heme through H-bonds with Tyr30 (B10) and Gln65 (E7) being responsible for the extremely low oxygen dissociation rate (0.11 s-1). A previous experiment showed that HbII is resistant to H2O2 and NO oxidation characteristics useful for the development of oxygen carrier derivatives. However, it is necessary to improve oxygen binding and induce allosteric properties of HbII. This was achieved through site-directed mutagenesis of HbII. Here, we report that recombinant HbII (rHbII) expression in bacteria was obtained after induction in a time course of 3--4 hours and the maximum rHbII protein yield obtained was 134.72 mg/L using the pET28 expression vector transformed in BLi5 cells. The optimal expression condition occurred when the induction was performed at 30 °C and when the terrific broth medium was supplemented with 1 mM IPTG and 60 microg/mL hemin chloride. To monitor the expression, we extracted the proteins and purified the rHbII using a metal resin. The protein product was analyzed using UV-Vis spectroscopy; the concentration of rHbII protein was determined using the molar extinction coefficient of 129 mM-1 cm-1 at 414 nm for the oxy-rHbII. Functional characterization of the rHbII was performed with the formation of oxy, deoxy, and CO complex. In addition, kinetics measurements of O2 affinity were performed using a Stopped-flow system and the dissociation rate constant value obtained was 0.0526 s-1, whereas the association rate constant was 0.19 x 106 M-1s -1. These values were similar to the native HbII dissociation and association constants, 0.11 s-1 and 0.390 x 10 6 M-1s-1, respectively. |
