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obtained at each protein concentration indicate that the protein does not selfassociate. The sedimentation coef cient (s0 ) was calculated as 7.08 0.09 S, 20;w from which the frictional ratio of SAT was found to be 1.53; this value is high when compared with the expected value for a globular protein ($ 1.15), supporting the hypothesis that SAT subunits are not organized in a compact motif. This hypothesis was con rmed using other techniques. The authors conclude that the fundamental building block is probably a trimer, with SAT arranged as a pair of loosely staked trimers. 13.9.2. Isolation of Retroviruses by Self-Generated Gradients18 Retroviruses are viruses in which the genetic material is RNA. Several retroviruses are related with human diseases, like in uenza or HIV. Some human retroviruses have glycoproteins as important surface structures; these are usually of crucial importance for infectivity, and therefore for virus characterization. Human cells from patients suffering from multiple sclerosis were cultivated, and the supernatants were separated from cells and debris after centrifugation at 1000 g at 4  C for 30 min. The supernatant was transferred to a 60-mL centrifuge tube, and underlaid with 4 mL of a concentrated solution of iodixanol, an iodinated, nonionic density gradient medium, in HEPES NaOH (60 mM, pH 7.4, 0.8% NaCl). This dense underlay medium is called a cushion , and prevents the pelleting of particles less dense. Particles then concentrate at the interface between the supernatant and the cushion. The tubes were centrifuged in a xed angle rotor (22.5 ) at 45,000 g (4  C, 2 h) and the supernatant removed from the tubes by aspiration, leaving only of 4 5 mL in the proximity of the cushion. The cushion and the overlying supernatant were combined, and the volume measured for nal regulation of the concentration of the gradient medium. This mixture, adjusted to contain 20% of iodixanol, was transferred to 11-mL tubes and centrifuged in a vertical rotor at 364,000 g (4  C, 3.5 h). Under these conditions, a gradient with a density from 1.02 to 1.25 was formed. Following centrifugation, the gradient was collected through a puncture in the tube bottom, in 0.5-mL fractions. Each fraction was assayed by PCR to determine retrovirus concentration. The authors showed that the iodixanol gradient medium is better for this purpose than the previously used sucrose medium. Sucrose gradient media have high viscosity and osmolarity when compared with iodinated gradient media; these properties can alter the external (glycoprotein) features of this virus, reducing infectivity; moreover, the gentle separations in iodixanol media allow the concentration of small amounts of virus in comparison with sucrose gradients. 13.9.3. Isolation of Lipoproteins from Human Plasma Lipoproteins transport lipids in blood; an early classi cation system was established using centrifugation methods, which are based on their different densities. Lipoproteins are classi ed as chylomicrons, largest and lowest in density
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(r < 0:96 g/mL), with low protein concentration (2%). Very low density lipoproteins (VLDL), low-density lipoproteins (LDL), and high-density lipoproteins (HDL) are progressively smaller, more dense and rich in protein, increasing the r and protein content up to 1.210 g/mL and 40 55% for HDL. Figure 13.1 shows the forces acting during centrifugation. If the particle r is lower than that of the centrifugation medium, the buoyant force becomes important and the particle moves upward. Techniques in which the particles to be analyzed move upward, reaching the surface of the centrifugation medium, are called otation or buoyant centrifugation separations. Standard centrifugation procedures used for fractionation of lipoproteins in human blood plasma are made by sequential otation using KBr, NaBr, NaCl, or a combination of these salts to produce a solution with determined density, in which a selected lipoprotein fraction moves upward. In a typical procedure, the human plasma is centrifuged (usually in a xed angle rotor) at 50,000 g for 30 min; under these conditions, chylomicrons oat as a milky layer. The infranatant is removed, and centrifuged at 100,000 g for 20 h, when a oating layer of VLDL is obtained, as well as a pellet, which contains the plasma proteins, LDL and HDL plasma lipoprotein fractions. The pellet is resuspended and mixed with a concentrated salt solution, in order to obtain a nal density of 1.063 g/ mL, and centrifuged at 100,000 g for 28 h. A oating layer of LDL is obtained, and the pellet is then resuspended and adjusted to a density of 1.21 g/mL. Centrifugation at 100,000 g for 48 h produces a oating layer of HDL. Using self-formed iodixanol gradients, a novel rapid method for the separation of plasma lipoprotein has recently been described.19 The centrifugation time is reduced to $4 h, but the gradient must be collected carefully, in 0.1 0.2-mL fractions. SUGGESTED REFERENCES
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P. Sheeler, Centrifugation in Biology and Medical Science, John Wiley & Sons, Inc., New York, 1981. D. Rickwood Ed., Centrifugation-A Practical Approach, IRL Press, Oxford, UK, 1984. J. Graham. Biological Centrifugation, BIOS Scienti c Publishers Limited, 2001.
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