Protein Chemistry - Part 1 - General Protein Chemistry - Ashmarin I. P. 1968

Determination of the size and shape of protein molecules
Comparative characteristics of methods used to estimate the size and shape of protein molecules

The Materials of this chapter show that the molecular weight and general characterization of the shape of a protein particle can be determined using a variety of Methods. If all conditions for their application are strictly observed and the Specific features of each method are taken into account, they yield very consistent results (see Table 5). Comparing the methods described above, however, it should be acknowledged that the most reliable molecular weight values are obtained by measuring the sedimentation velocity in an ultracentrifuge. The calculation actually utilizes not only ultracentrifugation data, but also the diffusion coefficient. This method (or, more precisely, the combination

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Fig. 41. Schematic representation of sedimentation diagrams for five successive stages of an experiment to determine molecular weight by the complete Sedimentation Equilibrium method (Schachman, 1959):

a — concentration change curves, b — concentration gradient change curves; 1–5 — curves from THE START OF the experiment until complete equilibrium is reached.

Table 5 Molecular weights of certain Proteins determined by various methods

of two methods) is advantageous because it simultaneously provides an estimate of the homogeneity of the test preparation. While not exhaustive, it significantly increases the researcher's confidence that the obtained molecular weight value corresponds to a sufficiently uniform fraction. Recently, newer and more practical modifications of the sedimentation equilibrium method have been increasingly used for molecular weight determinations, making it possible to study relatively low-molecular-weight proteins and to use relatively low-speed centrifuges. However, this method is unlikely to fully compete with the Sedimentation Velocity Method. A considerable amount of valuable data on molecular weight has also been obtained using the light scattering method. Nevertheless, it should be emphasized that this method is relatively less reliable, and values determined by it should ideally be verified using other methods. Results of molecular weight determinations obtained via osmometry, viscometry, and diffusion rate (without combination with ultracentrifugation) require even more cautious evaluation. The latter two methods are most valuable for a general Assessment of the shape—or, more precisely, the degree of Asymmetry—of the protein molecule. However, this requires that the molecular weight has been reliably established by other methods, such as ultracentrifugation.

It should not be forgotten, however, that none of the described methods can be considered ideal either for the precise Determination of molecular weight or, particularly, for assessing the shape of a protein molecule. In reality, protein molecules are neither spherical nor ellipsoidal, but have much more complex outlines that can only be approximated by these geometric figures. The true size and Shape of Protein particles can currently be assessed only through X-ray diffraction and Electron Cell/15.html">Microscopy, the principles of which are described in Chapter V. These techniques, especially X-Ray Structural Analysis, provide a complete picture of the quaternary and Tertiary Structure of A number of proteins, with General Data on molecular size and shape emerging as a secondary result. However, for the vast majority of proteins, the complete elucidation of their structure is still a long way off, as these methods are extremely complex and labor-intensive. Therefore, characterizing the size and shape of these proteins necessitates the methods described in this chapter.