Polymer Structure and Solubility of Deoxyhemoglobin S in the Presence of High Concentrations of Volume-excluding 70-kDa Dextran

Abstract

Earlier observations indicated that volume exclusion by admixed non-hemoglobin macromolecules lowered the polymer solubility ("C sat ") of deoxyhemoglobin (Hb) S, presumably by increasing its activity. In view of the potential usefulness of these observations for in vitro studies of sickling-related polymerization, we examined the ultrastructure, solubility behavior, and phase distributions of deoxygenated mixtures of Hb S with 70-kDa dextran, a relatively inert, low ionic strength space-filling macromolecule. Increasing admixture of dextran progressively lowered the C sat of deoxyHb S. With 12 g/dl dextran, a 5-fold decrease in apparent C sat ("dextran-C sat ") was obtained together with acceptable sensitivity and proportionality with the standard C sat when assessing the effects of non-S Hb admixtures (A, C, and F) or polymerization inhibitors (alkylureas or phenylalanine). The volume fraction of dextran excluding Hb was 70 -75% of total deoxyHb-dextran (12 g/dl) volumes. Electron microscopy showed polymer fibers and fiber-tocrystal transitions indistinguishable from those formed without dextran. Thus when Hb quantities are limited, as with genetically engineered recombinant Hbs or transgenic sickle mice, the dextran-C sat provides convenient and reliable screening of effects of Hb S modifications on polymerization under near-physiological conditions, avoiding problems of high ionic strength.The solubility of deoxyHb 1 S polymers at equilibrium with the tetrameric "monomers" is a basic thermodynamic property of the polymerization process that underlies red cell sickling. Measurement of the polymer solubility, "C sat "(1) (also termed C s (2) or C sol (3)), of Hb S in conditions similar to those within the cells has served as a critical estimate of the relative polymerization tendency when comparing the native or modified Hb S, alone or in mixtures with non-S Hbs or inhibitors of polymerization. A concentrated solution of Hb S is fully deoxygenated to form a gel, and after high speed centrifugation to sediment the polymerized solid phase, the concentration of soluble Hb in the supernatant is the measured C sat .It is well documented that volume exclusion by macromolecules admixed with proteins results in their increased activity and corresponding interactions (4). Thus, when protein macromolecules capable of interacting, such as deoxyHb S, occupy a substantial fraction of the total volume of their medium, the addition of a second species of noninteracting macromolecules crowds the protein molecules closer together, increasing their interactions. More specifically, addition of macromolecules that do not copolymerize with deoxyHb S reduces its polymer solubility by this mechanism (5, 6). However, for the use of such observations in further investigations on the properties of deoxyHb S polymers, it is first necessary to characterize the morphology and properties of the polymer fibers generated in volume-excluded domains by comparison with those previously characterized for standard conditions.In the course of our ...

How can biochemical reactions within cells differ from those in test tubes?

“…Any cell is extremely large relative to any particular macromolecule of interest and is likely to contain several micro-environments, within each of which a particular macromolecule X will be subject to a different set of background interactions. For example, the cytoplasm of even an organism as simple as E. coli contains at least three such micro-environments: the immediate vicinity of the inner plasma membrane, within which X will encounter a high local concentration of membrane phospholipids and proteins; the interior and immediate vicinity of the nucleoid, within which X will encounter an extremely high local concentration of DNA; and the remaining cytoplasm, within which X will be (Bookchin et al, 1999); large macromolecules (Minton, 1981;Nichol et al, increase in affinity of DNA-binding proteins for DNA (Zimmerman and Harrison, 1987;Jarvis et al, 1981). 1990); enhanced self-association of spectrin (Lindner and Ralston, 1995), actin (Lindner and Ralston, 1997), fibrinogen (Rivas et al, 1999), tubulin (Rivas et al, 1999) and FtsZ (Rivas et al, 2001).…”

A Role for the α113 (GH1) Amino Acid Residue in the Polymerization of Sickle Hemoglobin

“…We are now in a position to compare the individual strength of ␣113, ␣16, and ␣23 sites with other sites and also the interaction linkage of some fiber contacts in quantitative terms (Table III), because the C sat values of respective HbS mutants in all these cases have been determined in the presence of dextran under similar conditions, as described by Bookchin et al (30). The inhibitory strength of sites varies as follows: ␤95 Ͼ ␤88 Ͼ ␣113 ϳ ␣85 Ͼ ␣23 Ͼ ␣16.…”

“…Polymer Solubility of HbS Twin Peaks-The gelation concentration (C sat ) of HbS Twin Peaks was measured in the presence of high concentrations of dextran developed by Bookchin et al (30) and as described under "Materials and Methods." We carried out the polymerization of deoxygenated tetramers, HbS Twin peaks and native HbS, under identical conditions and subsequently measured the concentration of the respective hemoglobins in the supernatants to obtain their polymer solubility (Fig.…”

Direct intracellular measurement of deoxygenated hemoglobin S solubility

“…They can, of course, be added back, but accurate replication of intracellular conditions is difficult. Other methods requiring smaller amounts of hemoglobin are also in use, such as the high-phosphate method developed by Adachi and Asakura 3,4 and the dextran method developed by Bookchin et al 5 Although these methods have the advantage of consuming smaller amounts of hemoglobin, they deviate even further from physiological conditions and, particularly in the high-phosphate method, may systematically cause deviation from values obtained with the C SAT method. Another method requiring relatively small amounts of hemoglobin was developed by Benesch et al 6 In studies of HbS solutions, it was shown by Benesch et al 6 that the p50 is proportional to the amount of polymer formed because polymer has a much lower oxygen affinity than HbS tetramer.…”