Light-scattering investigation of the subunit dissociation of Homarus americanus hemocyanin. Effects of salts and ureas

Abstract

The subunit dissociation of the hemocyanin of the lobster, Homarus americanus, by the various salts of the Hofmeister series and the hydrophobic reagents of the urea-guanidinium chloride (GdmCl) class was investigated by laser light scattering molecular weight measurements. The dissociations of the hemocyanin dodecamers to hexamers by the various salts and the lower members of the urea series are found to be rapid and reversible, as predicted by the mass action law for monomer-dimer type of reactions. The salts are found to be very effective dissociating reagents with the unusual order of increasing effectiveness, Cl- less than Br- less than I- less than ClO4-, SCN-. The ureas and GdmCl are found to be relatively ineffective dissociating agents. In addition, the ureas show a decreasing order of effectiveness in going from urea to methyl-, ethyl-, and propylurea. This suggests that hydrophobic interactions are not the dominant stabilizing forces between the pairs of hexamers that form the dodecameric structure. Polar and ionic interactions appear to be the major stabilizing forces of the dodecameric structure. The use of equations derived for predicting the effects of dissociating reagents and salts on the structure of subunit proteins [Herskovits, T. T., & Ibanez, V. S. (1976) Biochemistry 15, 5715-5721] together with binding and Setschenow constants based on model amino acid data is found to give good account of the dissociation behavior observed with the salts, urea, and methylurea in the presence of calcium ion at both pH 7.8 and pH 9.5. The apparent number of amino acids at the contact areas of the hexamers, Napp, required to fit the dissociation data were found to be 24 +/- 8 at pH 7.8 and 23 +/- 4 at pH 9.5. However, because of the possible effects of molecular microheterogeneity, the estimates of amino acids at the contact areas must be viewed with caution, depending on further investigations.

Subunit structure and dissociation of Homarus americanus hemocyanin. Effects of salts and ureas on the acetylated and unmodified hexamers

“…* where RB is the measured Rayleigh ratio, K' is the lightscattering constant, and B'is the nonideality or second virial coefficient taken as 1 X lo-* L.mol.g-2 (Herskovits et al, 1981b).…”

“…The few hemocyanins that have been found that appear to be homogeneous or show only moderate effects of subunit heterogeneity are those of the isopods Ligia exotica and Bathynomus giganteus (Terwilliger et al, 1979;Van Holde & Brenowitz, 1981) and the lobster Homarus americanus (Morimoto & Kegeles, 1971; Herskovits et a]., 1981b). The association-dissociation behavior of the latter dodecameric protein to hexamers was found to be rapid and reversible, with the concentration dependence of the molecular weight correctly predicted by the law of mass action (Morimoto & Kegeles, 1971;Herskovits et al, 1981b).…”

Subunit‐association and functional properties in the hemocyanin of the squidtodarodes sagittatus

“…Figure 2 shows the relative populations of the different aggregation states of the molecule according to their dependence on pH (A) and on Ca +2 concentration (B). Differently from all the known similar systems (Siezen & Van Driel, 1974;Zolla et al, 1978;Bonaventura & Bonaventura, 1980;Van Holde & Brenowits, 1981;Van Holde & Miller, 1982;Brunori et al, 1985;Herskovits et a/., 1981), the '60 S species, i.e. the state of maximum aggregation, rapidly disappears in the presence of 10 mM CaCl 2 at pH values higher than 6.5, and an intermediate aggregation state (30 S) is stabilized.…”

High pressure stability of protein complexes studied by static and dynamic light scattering

“…particles. The measured intensity correlation function G (2) (τ ) can be related to the field correlation function G (1) (τ ) via the Siegert relation (2) .…”

“…Two hexamers built up the dodecameric hemocyanin of Homarus americanus. At physiological pH and ionic conditions, the dodecamer with a molecular weight of 940 kDa (estimated by ultracentrifugation) is the dominant species of the hemocyanin of H. americanus [1][2][3]. Dissociation studies were performed by means of urea, guanidinium chloride and salts of the Hofmeister series.…”