Ligand Binding Properties of Two Different Globin Domains and the Native Hemoglobin of Artemia Salina; A Comparison Study

Abstract

The brine shrimp Artemia expresses three hemoglobins (Hbs) - Hb I, Hb II, Hb III - which show different oxygen-binding characteristics. Genotypically, four globin chains (C1, C2, T1, and T2) are expressed. The proposed model for their quaternary structure is a heterodimer (Hb I: C1C2, Hb II: C1T2 and Hb III: T1T2) of two ring-shaped polymeric globin chains stacked coaxially. Each globin chain, (Mr ∼160.000) consists of a concatenation of nine globin domains (Mr ∼16.000) different in primary structure. In this study, domain 1 (AsHbD1) and domain 5 (AsHbD5) of chain C1 have been cloned, over-expressed and purified. The kinetic properties of the recombinant proteins as well as that of the native Hb (AsHbN) which was purified from the host were analyzed by using laser flash photolysis. The association rate constant (kon) of CO for the AsHbN as well as the recombinant proteins were measured by using the one pseudo-first order condition approach. The association constant value for AsHbD1, AsHbD5 and AsHbN were determined to be 3.32, 15.9 and 1.7 μM−1s−1, respectively. The nanosecond geminate rebinding constant (kgem) for all samples were measured and AsHbD5 showed higher kgem value. The fraction of ligand that does not escape the protein matrix (geminate fraction or Fgem) of domain 1 and domain 5 are very different, which in turn indicates a different heme pocket structure. In addition, the association and dissociation rate constants of O2 were measured by the displacement technique. It was shown that oxygen affinity (KO2) of AsHbD1 (1.34 μM−1) is higher than that of AsHbD5 (0.53 μM−1) whereas the KO2 of AsHbN is 0.36 μM−1. The results confirm different ligand binding properties of the two globin domains of Artemia Hb.