Structure and properties of hemocyanins. XIV. Reassociation of Helix pomatia alpha-hemocyanin

Helix pomatia α-hemocyanin dissociates within minutes into $\text{1}\text{10}$-size and $\text{1}\text{20}$-size molecules, on increase of pH in the alkaline region.The rate and final state of reassociation of $\text{1}\text{10}$-size and $\text{1}\text{20}$-size molecules have been studied, by turbidity measurements and ultracentrifugation, on lowering of pH or on the addition of calcium ions.Reassociation of $\text{1}\text{10}\text{-size}$ molecules proceeds in two phases, with half-times in the order of minutes and one hour, respectively. The slow phase is linked to the disappearance of transitory intermediates, presumably dimers and tetramers of $\text{1}\text{10}$-size molecules.A hysteresis is observed in the final state of pH-dependent and Ca²⁺-dependent dissociation-reassociation.Reassociation of $\text{1}\text{20}$-size molecules depends on the initial (isomeric) state of these molecules. The F(fast sedimenting)-$\text{1}\text{20}$-size molecules reassociate almost completely to whole molecules, whereas the S(slow sedimenting)-form does not reassociate at all.     

Structure and properties of hemocyanins. XII. Electron microscopy of dissociation products of Helix pomatia alpha-hemocyanin: quaternary structure

Electron microscopy of the dissociation products of α-hemocyanin of the snail Helix pomatia reveals two distinctly different conformations of $\text{1}\text{10}$-size molecules: a C(compact)-form at high ionic strength and pH near 8, and an L(loose)-form at low ionic strength and/or higher pH.The size and shape of the C-$\text{1}\text{10}$-size molecules indicate a dissociation of the $\text{1}\text{2}$-size molecules along the (10,9) helical grooves, which have been shown to be boundaries between the morphological units of the cylinder wall (Mellema &; Klug, 1972). The 5-fold collar is distributed evenly among the C-$\text{1}\text{10}$-size molecules.The C → L conformational change is characterized by a drastic loosening of the $\text{1}\text{10}$-size molecules to a flexible cluster of globules, with a concomitant decrease of sedimentation coefficient and increase of intrinsic viscosity and frictional ratio.The $\text{1}\text{20}$-size molecule appears as a flexible, linear chain of seven or eight globules with a diameter of 55 to 60 Å. These globules are inferred to be the minimal oxygen binding units with a molecular weight of about 50,000, linked together by short stretches of the polypeptide chain.Stable intermediate dissociation products, $\text{2}\text{10}$, $\text{3}\text{10}$ and $\text{4}\text{10}$-size molecules, were obtained by intramolecular crosslinking of $\text{1}\text{2}$-size molecules with dimethyl-suberimidate prior to dissociation.     

The dissociation of Helix pomatia alpha-hemocyanin. Microheterogeneity at pH 5.7 in 0.4 m NaCl.

The careful equilibrium light-scattering study by Engelborghs and Lontie (1973, J. Mol. Biol., 77, 577–587) of the dissociation into half molecules of the α-component of Helix pomatia hemocyanin demonstrated that, although complete dissociation was achieved in the presence of 1 m NaCl in 0. 1 m sodium acetate buffer at pH 5.7, incomplete dissociation occurred in 0.4 m NaCl in the same buffer in such a way that it could not be explained by simple application of the law of mass action. On the basis of their light-scattering data in acetate buffer containing 0.4 m NaCl, these authors indicated that the protein sample behaved like a mixture of thermodynamic species characterized by a multiplicity of dissociation constants. In this respect their conclusions were similar to those of Siezen and van Driel (1973, Biochim. Biophys. Acta, 295, 131–139) from their study of the influence of pH on association-dissociation processes at alkaline pH values. One simple model which actually fits the data of Engelborghs and Lontie reasonably well assumes the presence of some incompetent whole molecules, unable to dissociate into halves in 0.4 m NaCl: With a formation constant of 0.18 – 0.23 liters/g for whole molecules from half molecules, 30–35% of the material would be in the form of incompetent whole molecules. On the basis of this model, the major point of reinterpretation is that the sedimentation velocity diagram contains a fast Schlieren peak representing incompetent whole molecules and a slow peak representing a reaction mixture, rather than half molecules. This picture explains why the ratio of peak areas is independent of concentration, why material separated by ultracentrifugation appears not to reequilibrate when resubjected to ultracentrifugation, and why, on the other hand, mixing experiments have indicated rapid reequilibration of species.     

Relaxation kinetics of Helix pomatia alpha-hemocyanin. Whole-half molecule reaction at pH 5.7 in 0.4 M NaCl.

Two independent relaxation kinetics methods were used to study samples of α-hemocyanin kindly furnished to us by members of the Biochemical Laboratory of the University of Groningen. A Durrum-Gibson stopped-flow apparatus was used to obtain concentration-jump data in the light-scattering mode. A recently developed pressurejump light-scattering apparatus was used to obtain completely independent data. The studies were made in 0.1 m acetate buffer at pH 5.7 containing 0.4 m NaCl, conditions under which equilibrium light-scattering studies had been reported by Engelborghs and Lontie (1973, J. Mol. Biol., 77, 577–587). In the companion paper (Kegeles, 1977, Arch. Biochem. Biophys., 180, 530–536), a model is proposed, consisting of a system containing a mixture of reactive and unreactive whole molecules, from which data are derived for the formation constant of whole molecules from halves and the fraction of material which is capable of undergoing reaction. The present study uses this estimate of this fraction of reactive material to permit the evaluation of overall rate constants and equilibrium constants. When the estimate of 65% of reactive material derived without making nonideality corrections is applied to the kinetics data, very satisfactory agreement is obtained between the equilibrium constant acquired from equilibrium data and the equilibrium constants derived from the kinetics data.     

Binding of carbon monoxide to alpha-hemocyanin and beta-hemocyanin from Helix pomatia.

The binding of carbon monoxide to alpha and beta-hemocyanin from the snail Helix pomatia was studied under equilibrium conditions. Homotropic interactions upon carbon monoxide binding were much weaker than upon the binding of oxygen. Heterotropic interactions (Bohr effect and calcium-ion effect), however, were just as strong as in the case of the binding of oxygen. For alpha-hemocyanin a linkage has been observed between the binding of carbon monoxide and a change in quaternary structure of the protein.     

Models for the analytical ultracentrifuge behavior of Helix pomatia alpha-hemocyanin. I. Experimental testing of previous models

The 'microheterogeneity model' (R.J. Siezen and R. van Driel, Biochim. Biophys. Acta 295 (1973) 131) and the 'incompetent whole molecule model' (G. Kegeles, Arch. Biochem. Biophys. 180 (1977) 530) for the dissociation of Helix pomatia alpha-hemocyanin whole molecules to half molecules were tested experimentally, using ultracentrifugation and stopped-flow dilution analysis. Results of differential sedimentation experiments followed by stopped flow analysis of separated fractions of 60 S and 100 S molecules were not entirely as predicted by the incompetent model, the agreement depending on the pH and ionic strength of analysis. A considerable amount of stopped-flow dilution response could be attributed to material sedimenting between 60 and 100 S. This material appears to be the main equilibrating fraction, and its amount is considerably larger than that predicted by the microheterogeneity model. Increased hydrostatic pressure was found to enhance this fraction, whereas fixation or low ionic strength reduced or eliminated this fraction. Nonequilibrium components of 30, 50 and 80 S were detected and partially purified by differential sedimentation.     

Models for the analytical ultracentrifuge behavior of Helix pomatia alpha-hemocyanin. II. Simulations of reacting microheterogeneous hemocyanin

Simulations for the moving-boundary ultracentrifuge behavior of Helix pomatia alpha-hemocyanin have been developed, based on modification of published models. In the present treatment, it has been assumed that the protein system is extensively microheterogeneous, with respect to its whole-half molecule reactivity. It has also been assumed that all such association and dissociation reactions can proceed to equilibrium in a time appreciably shorter than that which would be required to separate nonreacting half molecules from nonreacting whole molecules. Predictions based on this model agree well with reported experimental findings of nonequilibration of fractionated material, apparent independence of whole-to-half molecule concentration ratios on total concentration in sedimentation experiments, and stopped-flow dilution reactivity over a wide range of sedimentation coefficients.     

Structure of Helix pomatia oxy-beta-hemocyanin and deoxy-beta-hemocyanin tubular polymers.

Mild trypsinolysis of Helix pomatia beta-hemocyanin leads to the formation of tubular polymers after removal of the collar part [van Breemen, J.F.L., Wichertjes, T., Muller, M.F.J., van Driel, R., and van Bruggen, E.F.J. (1975) Eur. J. Biochem. 60, 129--135]. Three-dimensional image reconstruction from electron micrographs of negatively stained tubular polymers showed: (a) alternating deep and shallow grooves in between the 10 helical chains, (b) the presence and position of two domains within each morphological wall-unit of the Mellema and Klug model [Mellema, J. E. and Klug, A. (1972) Nature (Lond.) 239, 146--150]. Optical diffraction of oxy and deoxygenated tubular polymers indicate a significant decrease in diameter with a concomitant increase in length upon deoxygenation.     

Dissociation of low density lipoprotein-antibody precipitates at alkaline pH

The effect of alkaline pH on the dissociation of immunoprecipitates of low density lipoproteins (LDL) of the S(f) 0-10 class was studied by immunological and ultracentrifugal methods. The precipitates prepared at the equivalence point were dissolved and centrifuged in sodium chloride solutions of density 1.063 and pH's between 10.25 and 11.5. Analytical centrifugation of the top fraction, which floated at density 1.063, after dialysis against 0.9% sodium chloride of pH 7.4 revealed the presence of LDL and of soluble LDL-antibody complex. The amount of soluble complex was greater for the preparations obtained at lower pH than those obtained at higher pH and was undetectable at pH 11.5. The yield of immunoglobulin from the bottom fractions was maximal when the pH of the centrifugation medium was 11.0. Below pH 11.0, the greatly reduced yield of immunoglobulin was due partly to incomplete dissociation and partly to aggregation of soluble complex, while above pH 11.0 the decreased yield was possibly due to alkaline denaturation of the globulin. The immunoglobulin separated at pH 11.0 and dialyzed to pH 7.4 was reprecipitatable by LDL, and the reactivity did not seem to be appreciably influenced by the alkaline treatment.     

Dissociation of Helix pomatia haemocyanin under the influence of alkali salts

The dissociation into halves of the α-component of Helix pomatia haemocyanin was investigated by light scattering and ultracentrifugation. Within the normal pH-stability region, a dissociation into halves can be obtained with alkali chlorides. This dissociation levels off at a salt concentration of 1 m. In contrast with the pH-induced dissociation, this process is both completely reversible and rapid.A study with different alkali halidea and other salts reveala the dissociation to be caused by the anions and not merely by an effect of the ionic strength. For some salts a dissociation into tenths and 20ths is found. The sequence of efficiency of the anions is practically that of the chaotropic ions (anions that favour the transfer of apolar groups to water). The effect of other parameters which influence the process is studied. The dependence on protein concentration is much smaller than expected from the law of mass action. This deviation, together with the unusual dissociation pattern of the protein in the ultracentrifuge, can be explained by the presence of different thermodynamic components.     

Proteolytic fragmentation of Helix pomatia alpha-hemocyanin: isolation of a functionally active chemically pure domain and evidence for subunit heterogeneity.

α-Hemocyanin of the vineyard snail, Helix pomatia, is a large oligomer composed of 20 subunits with a molecular weight of 360,000 ± 30,000. Limited proteolysis showed these subunits to be composed of about seven structural domains, each having one oxygen binding site (1). This paper describes the production of these structural domains by tryptic digestion of $\text{1}\text{10}$ hemocyanin molecules. The digestion pattern was followed as a function of time by examining the proteolytically obtained fragments electrophoretically in sodium dodecyl sulfate-containing polyacrylamide gels. Based on the molar ratio of each fragment present during digestion the first part of the reaction sequence for trypsinolysis could be deduced. This reaction scheme was simulated by means of an analog computer. Pseudo-first-order reaction rate constants of the various proteolytic cleavages were estimated from the computer generated time course of digestion. On the basis of these results it is postulated that Helix pomatia α-hemocyanin possesses at least two kinds of subunits which differ in their proteolytic susceptibility. These subunits occur in equimolar amounts. A functionally active domain with a molecular weight of about 50,000 has been isolated from a tryptic digest of hemocyanin subunits. This domain seems to be chemically pure, as suggested by the unique sequence of its first six amino acids, viz: Lys-Val-His-Leu-Asn-Lys.     

Primary structure of the low-molecular-weight carbohydrate chains of Helix pomatia alpha-hemocyanin. Xylose as a constituent of N-linked oligosaccharides in an animal glycoprotein

alpha-Hemocyanin of Helix pomatia is a copper-containing glycoprotein which serves as an oxygen carrier in the hemolymph. Its carbohydrate moiety has as constituents fucose, xylose, 3-O-methylgalactose, mannose, galactose, N-acetylgalactosamine, and N-acetyl-glucosamine residues. Alkaline borhydride did not split off any carbohydrate material, suggesting the absence of O-glycosidic chains. The N-glycosidic carbohydrate chains of this glycoprotein were liberated by hydrazinolysis of a Pronase digest then fractionated as alditols on Bio-Gel P-4. The fractions containing the low-molecular-weight glycans were investigated by 500-MHz 1H NMR spectroscopy in conjunction with sugar and methylation analysis. The largest, and most abundant, compound was established to be: (Formula: see text). Another compound was characterized as the afuco analogue of this structure. H. pomatia alpha-hemocyanin is the first example of an animal glycoprotein having xylose as a constituent of N-glycosidic carbohydrate chains.