Studies on the Subunit Structure of Four Arthropod Hemocyanins

Abstract

In view of the scarcity of information on the Southern hemisphere crustacean hemocyanin, further experiments have been carried out in this laboratory on Ovalipes catharus and Jasus edwardsii hemocyanins. This work also includes studies on the hemocyanins. Of another crab, Leptograpsus variegates, and the spiny lobster, Jasus novaehollandiae, found in waters off the Southern coast of Australia. We found the number heterogeneous subunits was seven for Ovalipes catharus, two for Jasus edwardsii and Jasus novaehollandiae, and four for Leptograpsus variegates hemocyanins. The molecular weight of these polypeptide chains ranged from 68,000 to 95,000, consistent with those of other arthropod hemocyanins. The dodecamers of these hemocyanins contained a greater number of heterogeneous subunits than the hexamers. The extra subunits present in the dodecamers may be the ‘linkers’ required to join two hexamers together to form a dodecamer. The absence of dimers in these crustacean hemocyanins suggests that inter-hexamer links are week, easily hydrolysed bonds. Hexamers that are incompetent to associate further are also present in the hemocyanins. Electron micrographs showed the hexamers as hexagonal, square or rectangular profiles, and the dodecamers as an association of two hexameric structures (a hexagon connected to a square or a rectangle) consistent with those reported for other arthropod hemocyanins. Reassociation of the subunits of Ovalipes catharus and Jasus edwardsii hemocyanins resulted in the formation of hexamers and dodecamers, some of which were different from those present in the hemolymph. Formation of the ‘correct’ dodecamers in reassociated Ovalipes catharus hemocyanin is less than 10% of those present in the hemolymph, which may be due to the incorrect reassembly of the highly heterogeneous subunits into hexamers which are incompetent to associate further, and the damage of some subunits by the alkaline conditions used for dissociation. The results also show that the heavy metals mercury, cadmium, copper, manganese and zinc affect the oxygen affinity and the co-operativity (at molar ratios of metal: protein between 2 and 6), as well as the spectral properties (at molar ratios of metal: protein between 2 and 16) of Ovalipes catharus and Jasus edwardsii hemocyanins. The binding sites for these heavy metals may be the sulphur bridges, trypotphans, tyrosines, histidines and the carboxylic groups of the amino acids either at or close to the active site of the hemocyanin. The number of heterogeneous subunits of Ovalipes catharus and Jasus edwardsii hemocyanins and their molecular weights differed from those reported by Robinson (1978), but are consistent with those of the Northern hemisphere hemocyanin. Similarly, the effects of the heavy metals on these two hemocyanins were similar to those of the Northern hemisphere arthropods.