Murex trunculus Haemocyanin

Abstract

The oxygenation of Murex trunculus haemocyanin was studied over a range of pH values and Mg2+ concentrations by following the increase in absorption a t 346 nm as oxygen was bound. I n the absence of Mg2+ the oxygen-binding curves appeared to be hyperbolic, n in the Hill plots was approximately unity, and there was no appreciable Bohr effect. I n the presence of Mg2+ the Hill plots showed a number of slopes and there was a marked, reverse Bohr effect. Deoxygenation of a solution haemocyanin at pH 9.9 in the presence of 0.01 M Mg2+, consisting of about 95O/, wholes and 5O/, halves, resulted in dissociation to 63.2 wholes and 36.8O/, halves, and this dissociation was slowly reversible upon re-oxygenation. Changes in oxygenation state were accompanied by changes in the circular dichroism spectrum. Upon deoxygenation, the bands a t 346, 480 and 560 nm of the copper * oxygen complex were abolished but there was little change in the doublet a t 210-222 nm. However, the positive band at -250 nm approximately doubled in intensity.I n the previous paper [l] some of the physical properties of the oxygen-binding protein haemocyanin from Murex trunculus were investigated; in all of these experiments the haemocyanin was in the fully oxygenated state. The native haemocyanin molecule contains about 160 pairs of copper atoms, that is 160 oxygen-binding sites, spread over a large but, a t present, uncertain number of subunits.It is of great interest to study the functional property of these giant molecules and to try to relate structure and function, especially as many haemocyanins, like haemoglobins, show sigmoidal oxygenbinding curves [2-41. As is well known [5] the binding of multiple ligands to equivalent sites on polymeric acceptor molecules in dynamic dissociation equilibrium can lead to sigmoidal binding curves and it would be surprising if the oxygen-binding behaviour of haemocyanin under a given set of conditions was not related to the conformation and arrangement of the subunits. Much interest has centred on the physical basis for sigmoidal binding curves since this lies a t the heart of our understanding of allosteric effects [6,7]. It has been suggested that the binding of small ligands to a multi-subunit protein will generally result in changes in intersubunit bonding energy. Ligand attachment results in structural alterations in the subunits and because the interactions between subunits are non-covalent and highly specific, this may result in dissociation or association [8]. It has already been shown [9,10] that the association state of two haemocyanins depends on the partial pressure of oxygen. The oxygenation reaction of haemocyanin is a complex system involving not only the protein and oxygen, but also the pH (Bohr effect) and the magnesium or calcium ion concentration. It is critically dependent on these parameters whether or not the so-called whole molecules (see [l]) dissociate or remain associated, and thus the potentiality for co-operativity is to a large extent controlled by them. It is unlikely that the m...

Oxygen-binding characteristics of three extra-cellular haemoglobins of Artemia salina

“…Finally, co-operativity in oxygenation of Artemia haemoglobin was dependent on the degree of oxygen saturation, the pH and the temperature, so agreeing with the properties of several other invertebrate haemoglobins (Waxman, 1971;Mangum & Carhart, 1972;Economides & Wells, 1975;Weber, 1975;Wood & Mosby, 1975;Bannister et al, 1976;Wood et al, 1976;Weber & Heidermann, 1977) and also in haemocyanins (Wood & Dalgleish, 1973;Pearson & Wood, 1974).…”

Functional Differences in the Multiple Hemocyanins of the Horseshoe Crab, Limulus polyphemus L

“…The oxygen-binding properties of many hemocyanins have been studied (17)(18)(19)(20)(21)(29)(30)(31)). It has not been possible previously, however, to make preparations which are electrophoretically homogeneous, nonaggregated and functional throughout the physiological pH range.…”

“…It has not been possible previously, however, to make preparations which are electrophoretically homogeneous, nonaggregated and functional throughout the physiological pH range. Functional heterogeneity of components in unfractionated hemocyanin preparations and variations in extent of aggregation must in part explain the heterogeneous binding curves observed for many hemocyanins (17)(18)(19)(20)(21). These complicating conditions seem to be absent in preparations of hemocyanin from Limulus which have been treated with EDTA and purified by ion-exchange chromatography (except that components III and V are not electrophoretically homogeneous).…”

“…Most hemocyanins have a positive Bohr effect, but reverse Bohr effects have been reported for hemocyanins from Limulus and, interestingly, from several gastropod molluscs (17)(18)(19)(20)(21)(28)(29)(30)(31). The presence of a reverse Bohr effect has been proposed to have physiological significance.…”

Morphological and functional correlates with distribution ofMurex trunculusL. andMurex brandarisL. (Mollusca, Gastropoda) in the northern Adriatic

“…In the northern Adriatic the two gastro- Dulzetto (1946Dulzetto ( , 1950 and Wondrak (1974), and the biochemistry of haemocyanin was the subject of several investigations (Bannister el al., 1975;Wood & Dalgleish, 1973;Wood & Peacocke, 1973).…”