Limuluspolyphemus hemocyanin is a 3.3 x 106-M, protein containing 48 subunits in an assemblage of eight hexamers. The molecule can be dissociated into monomers and dimers at pH 8.9 in the presence of 0.01 M EDTA. These subunits are heterogeneous and can be separated into five zones (I -V) by DEAE-Sephadex chromatography.Reassembly experiments were carried out with varied subunit mixtures, based on different combinations of the five chromatographic zones, in order to study the structural role of the diverse subunits in the eight-hexamer molecule. The reassembly products were analysed by electron microscopy and ultracentrifugation. No structural role for zone I could be found. Zone V and possibly zone I1 are needed to form structures larger than hexamers. Absence of zone I11 causes irregular aggregation of hexamers. Zone IV and perhaps zone I1 are needed to make eight-hexamer molecules from four-hexamer molecules. From these results we conclude that there is a high degree of subunit specificity in the inter-subunit contacts in the native Limulus hemocyanin molecule.Hemocyanins serve as oxygen carriers in the hemolymph of many invertebrates that belong to the phyla Mollusca and Arthropoda [ 1,2]. Hemocyanin molecules have discrete sizes depending on their biological origin. The quaternary structure of mollusc and arthropod hemocyanin molecules is very different. Changes in conditions, such as pH, ionic strength or concentration of divalent cations, causes hemocyanin molecules to dissociate into a number of distinct states of aggregation.Arthropod hemocyanins appear to be built from ensembles of one, two, four or eight hexameric units. The hexameric units have a sedimentation coefficient of about 16 S and a molecular weight of about 450000. At high pH, in the absence of divalent cations, these molecules dissociate into monomeric subunits with a sedimentation coefficient of about 5 S and a molecular weight of about 75000. Each 5-S monomer, consisting of one polypeptide chain, contains two copper ions and can bind one molecule of oxygen. Essentially all arthropod hemocyanin subunits which have been studied show heterogeneity in charge, molecular weight and sometimes in their functional properties [2-81.The hemocyanin in the hemolymph of the horseshoe crab Limulus polyphemus has a molecular weight of 3.3 x 10". It is an eight-hexamer molecule with a sedimentation coefficient of about 60 S. Dissociation into 5-S monomers occurs stepwise. The intermediate four-hexamer, two-hexamer and very rare one-hexamer structures, with sedimentation coefficients of 34 S, 24 S and 16 S respectively, are morphologically identical to undissociated hemocyanin molecules from other species when observed in the electron microscope (Fig. 1) [1,9]. The 5-S monomers are heterogeneous and can be separated by DEAE-Sephadex chromatography into five zones, called zones I-V [4]. Polyacrylamide gel electrophoresis shows the distribution of probably eight different polypeptide chains distributed over these zones [lo]. This study describes the structu...

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