Functional Differences in the Multiple Hemocyanins of the Horseshoe Crab, Limulus polyphemus L

Abstract

Hemocyanin in the hemolymph of the horseshoe crab, Limulus polyphemus L., is a high-molecular-weight copper protein which binds oxygen cooperatively and shows a higher oxygen affinity at pH 7 than at pH 9. Treatment with EDTA (ethylenediaminetetraacetate) disaggregates the hemocyanin molecules and abolishes both the reverse Bohr effect and cooperative oxygen binding. Chloride ions interact with the EDTAtreated material and, in the presence of saturating amounts qf NaCl, a reverse Bohr effect is restored, but cooperativity is not. The EDTA-treated hemocyanin contains at least five electrophoretically distinct hemocyanins. These hemocyanins have similar molecular weights (about 66,000) but are functionally dissimilar.They have different oxygen affinities and different responses to chloride ions. The effect of chloride ions on unfractionated hemocyanin is due to pH-dependent chloride interactions with only two of the five hemocyanin components. The functional differences between the hemocyanin components may provide Limulus with a valuable respiratory flexibility in its interaction with the environment. The kinetics of oxygen combination and dissociation for the various hemocyanin preparations show that variations in the rate of oxygen dissociation are primarily responsible for the observed differences in oxygen affinity. The rate of oxygen dissociation varies 20-fold under conditions where the apparent rate of oxygen combination shows less than a 2-fold variation. Cooperative interactions in the untreated hemocyanin are most obvious in the "off" reaction, which increases in rate as successive oxygen molecules are released.

Crystal structure of deoxygenated limulus polyphemus subunit II hemocyanin at 2.18 Å resolution: Clues for a mechanism for allosteric regulation

“…Even for Limulus hemocyanin the chloride effect is observed only for subunit types 11, IIA, IIIA, and IIIB. Detailed information on the regulation by chloride ions on both the native complex as well as on separate subunits can be found in Sullivan et al (1974), Brouwer et al (1982), and Brenowitz et al (1984). From these studies it becomes clear that the function of chloride ions is to reduce the oxygen affinity by more tightly binding to hemocyanin in the low-affinity state (T-state) than to the high-affinity state (R-state).…”

Magnetic susceptibility studies of laccase and oxyhemocyanin.

“…Limulus polyphemus hemolymph was strained through cheesecloth, centrifuged, and dialyzed against 50 mM Tris-glycine/10 mM EDTA, pH 8.9, and then against 50 mM Tris/10 mM EDTA, pH = 8.0. This procedure ensured that the oxyhemocyanin was disaggregated into a-mixture of subunits of molecular weight -70,000 (16). Copper concentration was determined by atomic absorption and by optical absorption (ew = 10,000 M-1 cm'1) (17) and found to be 4.70 i 0.05 mM (mean + SEM) by both methods.…”

Control of kinetics by cooperative interactions

“…Here, we want to focus on those cases where the speed of conformational transitions is deduced from the kinetics of oxygen dissociation when relaxing the system from full saturation (all in the R-state) to zero saturation (all in the T-state). The kinetics of oxygen dissociation of cooperative oxygen binding proteins is characterized by so called ''autocatalytic'' behavior (5)(6)(7)14). This means that the apparent off-rate of oxygen is not constant as it would be for a noncooperative homogenous systems but increases with time and thus with saturation degree.…”

“…This aspect is especially intriguing in case of invertebrate oxygen transport proteins because they occur often in very large assemblies and can have rather large Hill-coefficients (1)(2)(3). Of course, the number of parameters involved in the kinetic behavior of such assemblies is discouraging, nevertheless a couple of studies exist where the kinetics of oxygen binding and dissociation was investigated by stoppedflow and T-jump methods (4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17). In these experiments, some general properties were found: typically, high-and low-affinity states are much more similar in their rates for ligand binding than for ligand dissociation.…”