title: Static and dynamic light scattering approach to the hydration of hemoglobin and its supertetramers in the presence of osmolites.
abstract: We used static and dynamic light scattering for comparing the mass (MW) and hydrodynamic radius (R(h)) of several hemoglobin systems, namely human hemoglobin, bovine hemoglobin, human hemoglobin cross-linked with a sebacyl residue, and bovine hemoglobin cross-linked with an adipoyl residue. We measured the MW and R(h) of these systems in 0.1M phosphate buffer at pH 7.0 in the absence and in the presence of either betaine or glycerol up to 1.7 molal concentrations. The 90 degrees scattering was measured with a photon counting machine equipped with a diode laser at 783 nm. The Rayleigh ratio [R(theta)] of the instrument was estimated using R(theta) = 7.19E-6 cm(-1) for toluene at 783 nm. The refractive index increment of hemoglobin solutions was measured using a laser beam at 750 nm. We estimated a value dn/dc = 0.210 cm3/g in the absence and dn/dc = 0.170 in the presence of 1.7 molal osmolites. For all systems both in liganded and unliganded form, the static light scattering data showed a 16% mass increase with increasing concentration of osmolites. The hydrodynamic radii of all investigated systems in the presence and absence of osmolites were close to 3.17 nm. Assuming a partial specific volume nu = 0.739 for hemoglobin, and using spherical geometry, the estimated average hydration volume of hemoglobin was 32.6 L/mole in the absence of osmolites. It decreased to 23.5 L/mole in the presence of 1.7 molal osmolites. Assuming that the density of water in the hydration volume is D = 1.0 g/cm3, the hydration of Hb was 0.51 gH2O/gHb, with a surface density of 0.20 molH2O/A2. The hydration decreased to 0.33 gH2O/gHb and 0.14 molH2O/A2 in the presence of 1.7 molal osmolites. The decreased hydration was compensated by the increased mass (i.e., decreased surface area per unit volume) so that the thickness of the water shell around these proteins remained close to a single layer of water molecules. These findings indicate that the combination of static and dynamic light scattering offer unique means for investigating the relevance of water activity on the structure and function of biological macromolecules. In the case of hemoglobin, the data suggest that the decreased oxygen affinity in the presence of osmolites reported by Colombo et al. (M. F. Colombo, D. C. Rau, and V. A. Parsegian Science, 1992, Vol. 256, pp. 655-659), as due to ligand linked water binding on hemoglobin surface, is part of a complex phenomenon involving the hydration shell of hemoglobin and the formation of low affinity supertetrameric molecules.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Maryland Medical School, 108 N. Greene St., Baltimore MD 21201, USA.
authors.forename: Daniele
authors.initials: D
authors.lastname: Arosio
authors.forename: Herman E
authors.initials: HE
authors.lastname: Kwansa
authors.forename: Henry
authors.initials: H
authors.lastname: Gering
authors.forename: Grzegorz
authors.initials: G
authors.lastname: Piszczek
authors.forename: Enrico
authors.initials: E
authors.lastname: Bucci
mesh_terms: D000272:Adipates; D000818:Animals; D001622:Betaine; D001704:Biopolymers / Q000737:chemistry; D002417:Cattle; D003432:Cross-Linking Reagents; D003652:Decanoic Acids; D003998:Dicarboxylic Acids; D006441:Hemoglobin A / Q000737:chemistry; D006454:Hemoglobins / Q000737:chemistry*; D006801:Humans; D066298:In Vitro Techniques; D008027:Light; D008239:Lysine / Q000737:chemistry; D008970:Molecular Weight; D009994:Osmolar Concentration; D020836:Protein Structure, Quaternary; D021122:Protein Subunits; D012542:Scattering, Radiation; D012996:Solutions; D014867:Water / Q000737:chemistry
chemical_list: D000272:Adipates; D001704:Biopolymers; D003432:Cross-Linking Reagents; D003652:Decanoic Acids; D003998:Dicarboxylic Acids; D006454:Hemoglobins; D021122:Protein Subunits; D012996:Solutions; D014867:Water; D001622:Betaine; C029900:adipic acid; D006441:Hemoglobin A; C011107:sebacic acid; D008239:Lysine