DETERMINATION OF PROTEIN SUBUNIT COMPOSITION in Visual Studio .NET

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DETERMINATION OF PROTEIN SUBUNIT COMPOSITION
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Figure 10.2. Calibration curve for hemagglutinin molecular weight determination. [Reprinted, by permission, from D. J. Holme and H. Peck, Analytical Biochemistry , Longman, New York, 1998. # Addison Wesley Longman Limited 1998.]
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in measuring very small migration distances. The molecular weight of hemagglutinin was thus determined to be 160 kDa, which is within the linear region of the calibration curve. If the unknown protein showed a molecular weight in excess of $ 300 kDa, the experiment would be repeated using a lower range of %T values and a series of standard proteins of higher molecular weight. This technique yields molecular weight values that are accurate to within 5%. 10.3. DETERMINATION OF PROTEIN SUBUNIT COMPOSITION AND SUBUNIT MOLECULAR WEIGHTS2 Tertiary and quaternary structure in multisubunit proteins is maintained in part by the presence of disul de bonds between cysteine residues. When such a protein is exposed to a reducing agent such as mercaptoethanol or dithiothreitol, the disul de bonds are converted to sulfhydryl groups, the subunits dissociate and the tertiary structure of the subunits is disrupted. In the presence of SDS, such protein subunits yield uniformly negative species. Proteins will bind SDS in a 1.4 g SDS to 1-g protein ratio, effectively obliterating any native charge on the protein, and this uniform negative charge/mass ratio results in electrophoretic migration from cathode to anode with distances that depend only on the size of the polypeptide chain. The SDS polyacrylamide gel results in a simple molecular sieving action that allows small proteins to migrate rapidly, and restricts the migration of large proteins. Plots of log(relative molecular mass) (RMM, molecular weight) against relative mobility (migration distance) are linear, and allow the determination of unknown molecular weights. Figure 10.3 and Table 10.2 show data obtained for seven
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TABLE 10.2. Protein Standards Used for SDS PAGEa Number 1 2 3 4 5 6 7
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Protein Cytochrome c (muscle) Myoglobin (equine skeletal muscle) IgG Light chain Carbonic anhydrase (bovine) Ovalbumin Albumin (human) Transferrin (human)
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RMM 11,700 17,200 23,500 29,000 43,000 68,000 77,000
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log10RMM 4.068 4.236 4.371 4.462 4.634 4.832 4.886
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See Ref. 3.
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Figure 10.3. The SDS PAGE (a) and MW calibration (b) for proteins in Table 10.2.3 [Reprinted, with permission, from D. J. Holme and H. Peck, Analytical Biochemistry , Longman, New York, 1998. # Addison Wesley Longman Limited 1998.]
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MOLECULAR WEIGHT OF DNA BY AGAROSE GEL ELECTROPHORESIS
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proteins on a 5% polyacrylamide gel containing 0.1% SDS, after heating the samples in a mercaptoethanol SDS solution.3 10.4. MOLECULAR WEIGHT OF DNA BY AGAROSE GEL ELECTROPHORESIS4 The large pore sizes of agarose gels allow the molecular sieving effect to be employed for the determination of DNA molecular weight. Due to the negatively charged phosphate groups on the DNA backbone, native DNA has a constant charge/mass ratio. The relationship between molecular weight and mobility is not as straightforward for DNA as it is for protein MW determinations by SDS PAGE methods, however. Figure 10.4 shows plots of DNA length (in base pairs, n) against mobility, for linear and circular DNA. These plots show the general expected trend of decreasing mobility with increasing molecular weight, as well as decreasing mobility with increasing agarose content, or decreasing pore size. However, the semilogarithmic plot is nonlinear, especially at higher agarose concentrations, indicating that the simple sieving model does not strictly apply. The mobility behavior of DNA has been modeled using polystyrenesulfonate polymers.5 It has been shown that the mobility depends on the degree of entanglement of the polymer in the gel. Only weakly entangled polymers give the expected sieving behavior and linear calibration plots. This fundamental difference in mobility behavior between proteins and DNA is a result of the spherical, or random-coil, approximation that generally holds for proteins but does not hold for nucleic acid polymers. For DNA MW determinations, either the agarose concentration should be
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Figure 10.4. Mobility of linear ( ) and relaxed, circular (o) DNA through 0.3 or 0.6% agarose gels, at 0.3 V/cm for 30 or 40 h, respectively, in 0.1 M Tris, 0.09 M boric acid, and 1 mM EDTA.
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