Hemocyanin of the chambered Nautilus: Structure-function relationships

Abstract

The chambered nautilus, Nautilus pompilius, is a cephalopod mollusc that lives in deep water in the western Pacific. Structural and functional studies were undertaken on the high-molecular-weight copper protein that it uses as an oxygen carrier. Nautilus hemocyanin was found to resemble that of another cephalopod, the squid Loligo paelii. Unlike the hemocyanin of the gastropod molluscs, neither Nautilus nor Loligo hemocyanin exists in aggregation states with sedimentation coefficients of 100 S. The highest aggregation state in these two hemocyanins has a sedimentation coefficient of 58 S. This is significantly lower than the 64–66 S values found for half-dissociated molecules of typical gastropod hemocyanins. The dissociation of Nautilus hemocyanin was found to be sensitive to pH and divalent cations such as calcium and magnesium. At high pH, and in the absence of divalent cations, subunits that are believed to be large single polypeptide chains are found. They have sedimentation coefficients of 11 S, similar to subunit sedimentation behavior throughout the mollusc hemocyanin line. Unlike squid hemocyanin, Nautilus hemocyanin shows no intermediate dissociation products. Undissociated Nautilus hemocyanin shows a moderate oxygen affinity ( log P 1 2 = 0.8 at pH 8 ), a small Bohr effect ( log P 1 2 = 0.97 at pH 7.0 ), and a fairly low degree of cooperativity in oxygen binding ( n 1 2 = 1.5–2.0 ). Dissociation of Nautilus hemocyanin by dialysis against EDTA at high pH yields material of higher affinity, no cooperativity, and very slight pH sensitivity. The difference between the oxygen affinity of whole and dissociated Nautilus hemocyanin is most pronounced at low degrees of oxygen saturation. The question of possible subunit heterogeneity in Nautilus hemocyanin was addressed. Dissociated Nautilus hemocyanin was chromatographed at high pH in the absence of divalent cations. Three distinct peaks were found. These chromatographic zones, representing putative polypeptide chains of Nautilus hemocyanin, show significant differences in their oxygen binding characteristics. The presence of structurally and functionally diverse subunits in this cephalopod hemocyanin is reminiscent of that observed for two gastropod hemocyanins, and is thus suggestive that subunit heterogeneity may be a more general feature in the molluscan hemocyanins than previously appreciated.