Hemocyanins sedimentation

The data obtained on various proteins of moderate asymmetry are reported in Table 2. It should be noted that the zein preparations are highly polydispersed the others, while for the most part not strictly monodispersed, are fairly uniform, consisting chiefly of a single component, by ultracentrifugal and electrophoretic measurements, except for hemocyanin, which exists in three different sizes, as indicated. Sedimentation, diffusion and viscosity measurements have been carried out on all of these proteins and in general the agreement between the different methods is reasonabty satisfactory. 

Calcium causes aggregation of hemocyanin with an increase of sedimentation coefficient from 19S at 10 5M calcium to 100S at 10 2M (209). Gel filtration study of calcium binding indicates that each mole of hemocyanin (mol. wt 50,000 daltons) binds 20 moles of calcium (209). The association constant for this binding is 75 (209). No data are available to indicate the cation specificity of binding, but it seems safe to say that one can not consider this protein to be a calcium binding protein in any strict sense. 

Copper is an essential element, being active in many enzymes and hemocyanin. Copper is an essential nutrient element to animals and plants. However, high Cu accumulation in animals and plants can be toxic. Copper is found in three oxidation states including cupric (+2), cuprous (+1), and elemental Cu (0). Cu+ and Cu + are the most important forms and are involved in oxidation-reduction reactions in soils and sediments (Figure 12.7). Cu+ and Cu + can exist in aqueous systems, although the latter is much more dominant. Copper is widely distributed in nature in its elemental state and in the form of sulfide, arsenite, chloride, and carbonates. The earth s crust on an average contains approximately 50 ppm copper. Soil and sediment contain approximately... 

The introduction of the coefficient of viscosity measured by flow in the case where the particle moves in a pure liquid has been experimentally justified by Svedberg and Eriksson-Quensel (1936), who showed that the same sedimentation constant of Helix hemocyanin is obtained in mixtures of heavy and ordinary water in various proportions if corrections for density and viscosity are introduced. In the cases where the solvent is a dilute electrolyte solution, the necessity of a correction for the increased viscosity has not been completely proved. The corrections which are applied are in most cases sufficiently small to fall within the error of the determinations since for solutions of buffer below half-molar concentration the ratio i7bu o-/> watei does not exceed unity by more than 5%. 

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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