Biochemistry - The Chemical Reactions of Living Cells, Volume 1 - D. Metzler 1980

The Molecules of Which We Are Made
How We Study Molecular Structure
Determination of Molecular Weight

Determining the Molecular Weight of a biopolymer is often critically important. In many cases, a minimum molecular weight can be calculated from the content of its least abundant component (e.g., Tryptophan in a protein or iron in Hemoglobin). However, molecular weights are usually determined by Physicochemical Methods [151, 152]. In principle, this is quite straightforward to accomplish by measuring osmotic pressure and light scattering, though these methods also present certain challenges. Ultracentrifugation yields the most reliable results. Direct Determination of molecular weight is based on centrifuging a macromolecular solution until Sedimentation Equilibrium is established [125, 151]. When using short Cells, this equilibrium is reached within a few hours. The molecular weight is calculated by measuring The change in solution concentration from the center of The Cell to the bottom.

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FIG. 2-38. Dependence of the sedimentation coefficient of various Proteins and Nucleic Acids on molecular weight (logarithmic scale). Circles represent Globular proteins, triangles represent RNA, and squares represent DNA. The proteins include milk lipase, cytochrome c, pancreatic Ribonuclease, Lysozyme (from chicken egg white), follicle-stimulating hormone, bacterial proteases, human hemoglobin, bovine prothrombin, malate dehydrogenase, equine γ-globulin, tryptophanase (from E. coli), Glutamate dehydrogenase (from chicken), and cytochrome a. The double-stranded DNA is derived from the following sources: bacteriophage ΦX174 (replicative form), phages T7, λ_b2, T2, and T4, and papilloma virus. The RNAs include tRNA, rRNA, and mRNA from E. coli, and turnip yellow mosaic virus RNA (“CRC Handbook of Biochemistry”, Chem. Rubber publ. Co., Cleveland, Ohio, 1968; S. P. Colowick and N. O. Kaplan, eds., “Methods in Enzymology”, Vol. 12B, pp. 388–389. Academic Press, New York, 1968).

Most commonly, the molecular weight of Biopolymers is determined from their sedimentation coefficient s (Sec. 3.1.d). The value of s depends not only on the molecular weight but also on the density and shape of the molecule. However, assuming that protein molecules are spherical, s is approximately proportional to the 2/3 power of the molecular weight. Graphically, the relationship between logs and log (molecular weight) should appear as a straight line. Figure 2-38 shows a graph of this type, plotted using data for a wide range of proteins. Note that the points obtained for nucleic acids (which in many cases are rod-shaped rather than spherical) fall on a different straight line. Furthermore, the sedimentation coefficient changes more rapidly with increasing molecular weight than is typical for spherical molecules.

Currently, there are several novel Methods for determining molecular weight that can rival ultracentrifugation. One of these is simple Gel filtration. The Column is carefully packed with a gel (e.g., Sephadex) and calibrated by passing a series of protein solutions through it. One measures V_e, the volume of eluate collected from the moment the substance is applied to the column until its elution, and divides this volume by V₀, the elution volume for very large particles that do not penetrate the gel particles at all. Next, the dependence of V_e/V₀ on the logarithm of the molecular weight is plotted for a series of proteins with known molecular weights. As with the estimation of molecular weights from sedimentation constants, it is assumed here that the molecules of all proteins are roughly spherical in shape; for an unknown protein, the molecular weight is determined from THE POSITION OF its corresponding point on the aforementioned graph [153, 154]. A modification of this method is Chromatography at high concentrations of guanidine hydrochloride, a salt that induces Protein Denaturation. It is assumed that in such a solvent, the protein molecule behaves as a random coil [154].

Perhaps the most valuable new METHOD FOR DETERMINING molecular weight is gel Electrophoresis in the presence of the denaturing detergent sodium dodecyl sulfate. Under the action of this compound, proteins not only denature but also become coated with a more or less uniform layer of the detergent [155]. The resulting rod-like molecules typically exhibit a nearly uniform dependence of electrophoretic mobility on molecular weight. One such example is shown in Fig. 2-39, B. Here, too, the molecular weight of the studied protein is determined by comparing its migration rate with those of a series of proteins with known molecular weights [153, 156].

FIG. 2-39. A. Estimation of the molecular weight of azotioferredoxin in its "native" state using Sephadex G-200 gel filtration. Closed circles indicate the midpoint of the peak for proteins of known molecular weight. The arrow indicates the position of azotioferredoxin. The peak material for each protein was eluted into 5–7 tubes. The elution rate was 3.2 ml∙hr^-1. The eluate in the fraction collector was divided into 1.6 ml fractions. B. Estimation of the molecular weight of the azotioferredoxin polypeptide chain using sodium dodecyl sulfate-Polyacrylamide gel electrophoresis. Each data point was obtained from four standard curves. Marker proteins used were catalase (1), fumarase (2), aldolase (3), glyceraldehyde-3-phosphate dehydrogenase (4), α-chymotrypsinogen A (5), and Myoglobin (6). The open circle indicates the position of azotioferredoxin (Nakos G., Mortenson L., Biochemistry 10:457, 1971).