Chemistry and Biology of Proteins - F. Haurowitz 1953

Size and Shape of Protein Molecules
General Data

The Molecular Weight of low-molecular-weight substances can be determined by dissolving them in suitable Solvents and measuring the resulting depression of the freezing point, elevation of the boiling point, or reduction of the vapor pressure of the solvent. Satisfactory values are obtained using these Methods when the solute concentration is at least 0.01–0.1 M. For Proteins, however, these methods are inapplicable because it is practically impossible to prepare a 0.01 M protein solution with a molecular weight of 100,000. Such a solution would have to contain 1,000 g of protein per 1 L.

It is true that certain proteins, such as A number of albumins, are readily soluble, and it is not difficult to prepare solutions containing 300 g of protein per 1 L of Water or more. Even in these cases, however, it is difficult to use such solutions for determining protein molecular weights, as they typically contain trace amounts of low-molecular-weight contaminants that can drastically affect the results of the measurements. For instance, 1 mM carbonic acid and 1 mM serum albumin lower the freezing point of water to the exact same extent; meanwhile, the weight of 1 mM carbonic acid is merely 0.044 g, whereas the weight of 1 mM serum albumin is 68 g.

Errors of this kind, caused by the presence of impurities, can also occur in experiments where the molecular weight of proteins is determined from the boiling point elevation or the solvent vapor pressure depression. From what has been said, it is clear that determining the molecular weight of high-molecular-weight compounds like proteins requires The Use of other methods.

The earliest determinations of protein molecular weights were based on the chemical quantification of elements or Amino Acids present in trace amounts within the protein. A classic example of this approach is the determination of Hemoglobin molecular weight based on its iron content. Mammalian hemoglobin contains 0.34 g of iron per 100 g of protein. The atomic weight of iron is 56, and therefore 0.34 g equals 0.34/56 = 1/165 eq; hence, The amount of hemoglobin bound to 1 g-eq of iron is 165∙100 = 16,500. This value, representing the minimum molecular weight of hemoglobin, was long regarded as its true molecular weight. It was later established that the true molecular weight of hemoglobin in solution is 68,000, leading to the Conclusion that each hemoglobin molecule actually consists of four smaller units, whose molecular weight was precisely that determined from the iron content.

In the same manner, using the content of Certain amino acids, the minimum molecular weights of ß-lactoglobulin, serum albumin [1], edestin [2], and other proteins were calculated. Although such chemical analysis does not yield true molecular weights but only indicates their minimum values, it can nonetheless prove highly useful, especially when different determinations (based on various constituent PARTS OF THE molecule) yield the same minimum weight value.

The Physicochemical methods used to determine protein molecular weights are based on different principles and sometimes yield widely divergent results, the interpretation of which is often difficult and occasionally impossible. This is because measurement outcomes depend not only on the size and mass of the protein molecules, but also on their electrical charge and shape. The latter factor is particularly significant when determining the velocity of molecular movement, such as The rate of diffusion or sedimentation in a gravitational field. While globular molecules behave predictably in such experiments, elongated, rod-like molecules of Fibrous proteins exhibit anomalous behavior. Deviation from a spherical shape leads to an increased frictional coefficient and, consequently, a decreased rate of diffusion. Measurements in concentrated solutions containing thread-like molecules present additional complications arising from mutual collisions and temporary associations between molecules. Results obtained by dynamic methods are also affected by particle Hydration, as the movement of molecules through the solvent is slowed down if their effective diameter increases due to hydration.

The complicating factors mentioned above do not, however, pose a major hazard when investigating protein solutions at equilibrium, as is the case, for example, when measuring osmotic pressure or determining the concentration gradient in the gravitational field of an ultracentrifuge. In the following sections, we will first discuss these equilibrium methods, then examine dynamic methods, and finally consider some other approaches to determining protein molecular weights.