Yingtai: Determining Molecular Mass And Sedimentation Coefficient During Ultracentrifugation

Yingtai: Determining Molecular Mass and Sedimentation Coefficient During Ultracentrifugation

Ultracentrifugation is a crucial experimental technique for separating and analyzing biological macromolecules such as proteins and nucleic acids. Determining the molecular mass and sedimentation coefficient of these molecules is essential for understanding their properties.

Determining Molecular Mass:

1. Sedimentation Equilibrium Method:

   - In this method, the sample is placed in a strong centrifugal field, and molecular mass is determined using sedimentation equilibrium. By measuring the concentration distribution at different positions in the sedimentation cell, the mass of individual particles or the molecular weight of polymers can be calculated. When diffusion and sedimentation reach equilibrium, the molecular mass can be determined using the concentration measurements from various positions in the sedimentation cell. The formula involves parameters such as the Boltzmann constant, gas constant, thermodynamic temperature, partial specific volume of the particle, density of the medium, rotational angular velocity, and the distance from the rotational axis in the sedimentation cell.

2. Sedimentation Velocity Method:

   - If the centrifugal field strength is increased to 10^5 to 10^6 times the gravitational field, diffusion effects can be neglected. Under these conditions, particles move under the influence of centrifugal force. The sedimentation coefficient can be determined by measuring the position of the interface at different times. By monitoring the position changes of particles in the sedimentation cell over time, the sedimentation coefficient can be calculated, which indirectly reflects the molecular mass.

Determining Sedimentation Coefficient:

1.Definition of Sedimentation Coefficient:

- The sedimentation coefficient (S) is defined as the rate of sedimentation of a particle in a unit centrifugal acceleration field. It is an important parameter reflecting the size and shape of the molecule. The calculation formula is S = v / (ω^2r), where v is the sedimentation velocity, ω is the angular velocity of the centrifuge rotor, and r is the distance from the center of rotation.

2.Specific Measurement Method:

   - Using an ultracentrifuge, the sedimentation coefficient is determined by observing the movement of particles in the centrifugal field. Different molecules have different sedimentation velocities due to variations in mass, density, and shape. Typically, the sedimentation coefficients of proteins and nucleic acids range from 4S to 40S, ribosomes and their subunits range from 30S to 80S, and polysomes exceed 100S.

In summary, through the sedimentation equilibrium or sedimentation velocity methods in ultracentrifugation, one can effectively determine molecular mass and sedimentation coefficients. These methods not only provide fundamental insights into molecular mass but also help scientists better understand molecular interactions and functions.