Fundamentals of Biochemistry - Filippovich, Y. B. 1999

Proteins
Molecular Weight of Proteins

The molecular weight of a protein is measured using various physical Methods, including gravimetry, viscometry, osmometry, ultrafiltration, Gel filtration, Electrophoresis, and optical methods. The resulting value is commonly referred to as the physical molecular weight of the protein; it is determined with an accuracy of a few hundred, and sometimes thousands, of daltons. The Introduction/19.html">Primary Structure of many Proteins has now been elucidated, making it possible to calculate the molecular weight with an accuracy down to hundredths of a dalton, thereby yielding the chemical molecular weight of the protein. Usually, researchers deal with the physical molecular weights of proteins, which vary across a very wide range for the entire spectrum of native proteins (Table 3).

Class="center">Table 3 Molecular weight, degree of molecular Asymmetry, and isoelectric points of certain proteins

The Determination of protein molecular weight by the gravimetric method is performed in analytical ultracentrifuges (Fig. 15). THE PRINCIPLE OF the quantitative study of suspended particle sizes in a centrifugal field was put forward by A. V. Dumansky as early as 1913, and he also attempted to determine particle sizes based on the sedimentation rate in conventional laboratory centrifuges. However, the first ultracentrifuge equipped with an optical attachment that allowed observation and photography of the particle sedimentation process was built by the Swedish physical chemist T. Svedberg ten years later. During the rotation of its rotor, the centrifugal acceleration developed was only 150 times greater than the acceleration due to gravity.

In modern standard ultracentrifuges, this ratio reaches 300,000, and in specialized ones, it reaches 900,000–1,200,000. This value is called the relative centrifugal force or Separation factor: Ф_р = ω^2r/g, where ω is the angular velocity of the rotor, rad/s; r is the distance from the center of the rotor to the middle of The Cell containing the protein solution, cm; g is the acceleration due to gravity, cm/s². Thus, for example, the notation 100,000 g means that the centrifugal acceleration at the Location OF THE cell within the ultracentrifuge rotor exceeds the acceleration due to gravity by 100,000 times. The separation factor achieved in an ultracentrifuge is one of its most important characteristics.

Fig. 15. STRUCTURE OF THE ultracentrifuge:

1 and 2 — upper and lower steel plates; 3 — wall of the steel cylinder; 4 — steel needle with a suspended rotor (II); S — rotor drive; 6 — thermocouple; 7 — mounting plate; 8 — electromagnetic shutter; 9 — metal pipe connecting the vacuum chamber to the camera; 10, 13 — upper and lower quartz windows; 12 — steel rotor tail in the guide bushing; 14 — rotor housing for the cell used to observe protein particle sedimentation

The protein solution under study is placed in a small, light-transmitting cell, which is inserted into a specialized rotor housing. Under the centrifugal force generated by the Rotation of the rotor, the protein molecules in this cell gradually settle to the bottom. An optical attachment to the ultracentrifuge makes it possible to take photographs of the cell's contents at regular intervals, thereby determining the Sedimentation Rate of the protein particles. The molecular weight of a protein can be determined by ultracentrifugation using two approaches: based on the sedimentation velocity of protein molecules and based on Sedimentation Equilibrium.

In the first approach, The rate of movement of the solvent-protein boundary within the ultracentrifuge cell is measured and related to the centrifugal acceleration applied, yielding the sedimentation coefficient: s = v/ω^2r, where V is the velocity of the solvent-protein boundary movement, cm/s, and ω^2r is the centrifugal acceleration, cm/s². The dimension of s is expressed in seconds. A sedimentation coefficient of 10^-13 s is taken as a unit and named the svedberg (S). By substituting the experimentally determined sedimentation coefficient into the formula, the molecular weight of the protein is calculated:

where R is the gas constant; T is the Temperature (in Kelvin); D is the diffusion coefficient; ρ is the density of the solvent; σ is the density of the protein particles.

In the second approach, the protein concentrations c₁ and c₂ are measured at two points in the cell at distances x₁ and x₂ from the center of the rotor at the moment when, after a specified period of ultracentrifugation, sedimentation equilibrium is established in the cell—that is, equality between the number of sedimenting protein molecules and those diffusing in the opposite direction. The calculation of the protein's molecular weight is performed using the formula by substituting the experimentally found values of c₁, c₂, x₁, and x₂:

where to is the angular velocity, and the remaining designations are the same as in the previous formula.

Among the remaining physical Methods for determining protein molecular weights listed above, two more are widely used: gel filtration and electrophoresis.

The first method consists either in determining the volume of eluent required to elute the protein from a Sephadex gel Column (V_e) and relating it to the void volume of the column (V₀), or in establishing the migration distance of the protein in a thin layer of Sephadex on a plate. Both of these parameters are functionally related to the molecular weight of the protein. Using calibration curves constructed from V_e/V₀ or from the migration distances of marker proteins (i.e., proteins with known molecular weights), the molecular weight of the test protein is found.

The second method involves measuring the distance migrated by the protein during Polyacrylamide gel electrophoresis; here, too, a relationship exists between the molecular weight of the protein and the migration distance. This relationship becomes even more pronounced when comparing the retardation of protein migration during electrophoresis in a gel with a lower acrylamide content to electrophoresis in a gel with a higher acrylamide content. In this case, calibration curves are also constructed using marker proteins to determine the molecular weight of the unknown protein.

Other physical methods for determining the molecular weights of proteins—such as light scattering, viscosity, and Osmotic Pressure of Protein Solutions, as well as X-Ray Diffraction Analysis and Electron Microscopy—are used extremely rarely.

The chemical method enjoys some application. Its essence lies in the Quantitative determination of an element or amino acid within the protein composition that is present in the smallest amount. A calculation of the minimum molecular weight is then carried out, based on the assumption that a protein molecule cannot contain fewer than one atom of an element or one amino acid residue. However, this method does not always provide an accurate representation of the true molecular weight of a protein. For example, hemoglobin (a protein found in the Blood of humans and animals) contains 0.34% iron. Since hemoglobin cannot contain less than one Fe atom, the minimum molecular weight of hemoglobin is calculated proportionally: 0.34 parts of Fe correspond to 100 parts of protein, 56 parts of Fe (one atom) correspond to x parts of protein; hence, x = (56 ∙ 100) / 0.34 = 16,500. The true molecular weight of hemoglobin is 66,000–68,000, which is four times the minimum molecular weight. This is natural, since a hemoglobin molecule contains 4 Fe atoms. Thus, the chemical METHOD FOR DETERMINING the molecular weights of proteins is to a certain extent conventional.