PRELIMINARY CRYSTALLOGRAPHIC DATA OF SUBUNIT III AND SUBUNIT IV OF HEMOCYANIN FROM LIMULUS POLYPHEMUS. Karen A. Magnus and Shenping Liu. Depart. Biochem., Case Western Reserve Univ. School of Medicine, Cleveland, Ohio 44106, USA.

Hemocyanins are oxygen carriers with dicopper oxygen binding sites. Their basic assembly units are hexamers. The hemocyanin from Limulus polyphemus has 8 distinct subunits which form a 48mer as the biological active molecule. These subunits have different oxygen affinities, different cooperative phenomena and different assemblely characteristics. Subunit IIIa is the only subunit in Limulus hemocyanin which can catalyze the decomposition of H₂O₂ to O₂ and H₂O. Subunit IV is the only subunit which assembles into hexamer under low ion strength in the presence of Ca₂₊ ion. We crystallized these two hemocyanin subunits under conditions favoring hexamization using hanging drop vapor diffusion methods. For subunit III, we use 8.5-9.0% PEG3350 as the precipitant in 0.3M sodium cacodylate, 1M NaCl, pH=6.2-6.4. Crystals appear within 2 weeks and grow to 0.8*0.5*0.5 mm in abouout 90 days at 4deg.C. At room temperature they show 6Å difrraction with our in-house X-ray generator. The space group is C222(1) with a=218.21, b=368.43, c=237.86, a=b=g=90.0 . Assuming 2 hexamers/per asymmetric unit, the Vm is 2.6Å?/Da. Subunit IV crystals appear in several days and reach their full sizes within two weeks under the following conditions: 6-9%PEG3350, 0.2M Bis-tris, 0.22MCaCl₂, pH=4.9-5.4, at 4deg.C. Crystals diffract at least 3Å at room temperature. They are an orthorombic space group with a=106.84 b=127.41, c=346.37, a=b=g=90.0. With a hexamer /per assymetry unit, it gives a Vm=2.6Å?/Da. NSF MCB 9305250