Studies on Ligand Binding to Hemoglobins from Teleosts and Elasmobranchs

Abstract

Abstract Hemoglobins from three sharks, the porbeagle (Lamna nasus), the dusky (Carcharhinus obscurus), and the mako (Isurus oxyrinchus), and four bony fishes, the big-eye tuna (Thunnus obesus), the swordfish (Xiphias gladius), the carp (Cyprius carpio), and the smallmouth bass (Micropetrus dolomieu), have been examined by a variety of techniques to determine both their molecular structure and their kinetic and equilibrium ligand-binding properties. For all species multiple hemoglobins were found in the hemolysates. These hemoglobins are tetrameric and exhibit cooperative ligand binding. The main components of the hemolysates from the oceanic species consist of either three or four electrophoretically separable globin chains. However, the hemolysates behave as if they contain only two distinct functional components which appear to be present within the same tetrameric molecule. These two types of ligand-binding sites exhibit different kinetic and spectral properties for the carbon monoxide-binding reaction and for the displacement of oxygen by carbon monoxide. At 20° the differences between the rates of oxygen dissociation from these two components are largest for the hemoglobins of the mako shark (22.7 s-1 and 1.86 s-1) and those of the swordfish (28.2 s-1 and 4.8 s-1). Similar albeit smaller differences were observed for porbeagle, dusky, big-eye tuna, and carp hemolysates. The carbon monoxide-binding reactions of these hemolysates were also separable into a fast and slow component with all species showing a rapid reaction on the short wave length side of the 425 nm isosbestic point and a slower reaction on the long wave length side. With the possible exception of big-eye tuna hemoglobin, all of the fish and shark hemoglobins appear to interact with organic phosphates with a stoichiometry of 1 mole bound per mole of hemoglobin tetramer. At neutral pH inositol hexaphosphate lowers the rate of CO binding to swordfish and bass hemoglobins by a factor of 10 to 20 but appears to affect only half of the heme sites. Although less dramatically, this organic phosphate also lowers the rate of CO binding to the shark hemoglobins. The oxygen equilibrium curves of porbeagle and big-eye tuna hemoglobins change shape with temperature, but the concentration of oxygen at 50% saturation for the former is independent of temperature and that of the latter varies only slightly in going from 35 to 5°. The basis of this unusually small temperature dependence lies in a differential temperature response of the two types of functional components which appear to be present within the same tetramer. For big-eye tuna hemoglobin, the rate of oxygen dissociation for one of the components is the same at 3° as it is at 20°, approximately 50 s-1. For porbeagle hemoglobin, the rate of ligand association to one of the components present in the deoxygenated hemolysate is 36 times smaller at 3° than at 20°. Both of these responses produce unusually low affinity species at 3°.