Functional differences in the multiple hemocyanins of the horseshoe crab, Limulus polyphemus L.
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 (ethylenediaminetetra-acetate) disaggregates the hemocyanin molecules and abolishes both the reverse Bohr effect and cooperative oxygen binding. Chloride ions interact with the EDTA-treated material and, in the presence of saturating amounts of 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.
D000818:Animals; D003386:Brachyura / Q000378:metabolism*; D002712:Chlorides / Q000494:pharmacology; D002850:Chromatography, Gel; D004492:Edetic Acid / Q000494:pharmacology; D006433:Hemocyanins / Q000032:analysis / Q000378:metabolism*; D006458:Hemolymph / Q000032:analysis; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010100:Oxygen / Q000097:blood*; D011485:Protein Binding / Q000187:drug effects; D012965:Sodium Chloride / Q000494:pharmacology
D002712:Chlorides; D012965:Sodium Chloride; D006433:Hemocyanins; D004492:Edetic Acid; D010100:Oxygen