Hemocyanin stability

Both cations play a stabilizing role in various biological structures. Calcium ions are necessary to link the 24-subunit aggregates of the dioxygen carrier hemocyanin of Limulus polyphemus into the native 48-subunit molecule.121 The divalent character of Ca2+ probably allows it to crosslink... 

Solomon et alf° have suggested that the peroxide is bound to a single copper at the binuclear type 3 site, and so diflers from the bridging peroxide found for hemocyanin and tyrosinase (Figure 57). Comparison with a laccase from which the type 2 copper has been removed has led to the suggestion that the type 2 copper is necessary to stabilize the type 3 Cu-hydroperoxide complex. The type 2 and type 3 centres appear to be close together, as rapid-quench ESR experiments with reduced laccase and have shown that the thus formed is bound equatorially at the... 

The stability of the black copper complex and its tendency to form soluble, highly colored complexes with amines (and also denatured albumen) indicates that the compound we have in hand is involved in the union of the copper to the protein in hemocyanin. The function of the prosthetic group thus appears to be the same as that of protoporphyrin in hemoglobin, namely, to provide a basis for a very stable metallic complex. Beyond this analogy, however, there seems to be little or no chemical relationship between the prosthetic groups in limulus hemocyanin and hemoglobin. It should be noted that since limulus hemocyanin differs markedly in its copper content from the hemocyanin of other species,2 the conclusions we have drawn do not necessarily apply to the nature of the prosthetic group in the other hemocyanins. 

Comparing the most conserved parts, the active site, reveals two different types of type 3 copper proteins, an arthropod hemocyanin-like one and a molluscan hemocyanin-like one. While the Cu-B site is highly conserved in both types, the Cu-A site differs. In the case of molluscan hemocyanins, one helix is too short for stabilizing the important histidine complexing Cu-A so that it has to be tied down by an unusual His-Cys bond as shown in Figure 2 (d-f). 

Biomimetic copper-dioxygen chemistry has advanced considerably since the first structurally-characterized copper-dioxgygen adduct. However, it has been difficult to simulate the room-temperature stability of hemocyanin in these model complexes due to the fact that unlike the enzyme active sites, these models usually do not possess protective environments which can help stabilize potentially reactive copper-dioxygen species. Recently, two room-temperature stable copper-dioxygen complexes have been synthesized which come closer to the goal of mimicking the dioxygen carrier hemocyanin. 

As an important consequence of resonance stabilization there will be low-lying charge transfer bands in the absorption spectrum. Hemocyanin has a molar extinction coefficient Cmax = 750. Laccase of mammalian plasma has an absorption band at 6050 A with. e,nax = 1200. We may wonder if those bands are really charge transfer bands. On the other hand we must note that the theory of ESR spectra is still in its infancy and that we should be cautious in our interpretation. 

The stability of hemocyanin above a certain pH value is extended by the presence of calcium or magnesium ions. In a 0.01 M solution of calcium chloride, dissociation of hemocyanin from Helix pomatia does not begin before pH 9.3 and whole molecules are still present at pH 11-11.5 when, under normal conditions, irreversible splitting of the molecule would have occurred (Brosteaux, 1937 Putzeys and Van de Walk, 1939 Brohult, 1949). 

The dissociation of Helix pomatia hemocyanin has been the object of study for many years by the school of Louvaine (See Lontie, 1957 for references). Dissociation of the particles into halves occurs to a maximum extent of 75% on addition of sodium chloride to a solution of hemocyanin. By preparative ultracentrifugation it was possible to separate the dissociated molecules from a sediment of undissociated particles. Since the ratio of both hemocyanins remained constant under several different experimental conditions, the hypothesis was formed that two components are present in the respiratory pigment of Helix pomatia a-hemocyanin, which in the stability region dissociates into halves in the presence of sodium or potassium chloride, and /3-hemocyanin, which does not dissociate under these conditions (Lontie, 1954, 1957). Recently, these components were separated and purified ot-hemocyanin has been isolated by preparative ultracentrifugation whereas j8-hemo-cyanin was crystallized by dial)rsis at a pH near the isoelectric point (5.3). Study of the absorption spectra showed that, whereas the copper bands of the p component are constant, those of ochemocyanin vary from one preparation to another. The absorption coefficients in the copper hands of a-hemocyanin are also very variable and always lower than those obtained for j3-hemocyanin (Heirwegh and Lontie, 1960 Heirwegh et al, 1961). 

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