Protein Chemistry. Structure, Properties, Research Methods - Shendryk A.N. 2022

Methods for Experimental Study of Protein Structure
Methods for Determining Protein Molecular Weight
Determination of Molecular Weight by Sedimentation - Approach to Sedimentation Equilibrium Method

This method represents a compromise between the two approaches discussed above. It lacks the inherent precision of Sedimentation Equilibrium, but offers a substantial reduction in measurement time. While sedimentation equilibrium demands precise measurement and Maintenance of the rotor speed, the approach-to-equilibrium method brings the rotor speed to equilibrium within 1 to 2 hours through a series of successive adjustments. Each time, the protein concentration at the bottom of The Cell is measured, and the protein molecular weight is determined from these data by extrapolation.

Density gradient centrifugation. This method is widely used not only for determining sedimentation coefficients, but also for separating mixtures of macromolecules, Organelles, and Viruses. There are two variants of density gradient centrifugation. In one, a sucrose density gradient is established in the centrifuge tube. This is achieved by using automatic pumps to mix a concentrated sucrose solution with Water in gradually decreasing ratios. The mixture of macromolecules dissolved in a light solvent is layered on top of the gradient solution. During centrifugation in a swinging-bucket rotor, macromolecules of each type sediment along the density gradient at a characteristic rate, which is primarily determined by particle mass. This sedimentation process forms distinct bands or zones, enabling rapid and effective Separation. The sedimentation coefficient of a protein is determined from THE POSITION OF its band in the gradient relative to the positions of marker protein bands with known sedimentation coefficients.

The second modification of this method is not used for molecular weight determination; however, it is highly effective for separating macromolecules with different densities. In this approach, a concentration gradient is not pre-formed. Instead, a 6M CsCl solution serves as the medium, characterized by high solubility and low viscosity at high concentrations. In a strong gravitational field, CsCl itself undergoes slight sedimentation, resulting in The formation of a concentration gradient. Establishing this gradient typically requires a considerable amount of time, ranging from several hours to several days. The density distribution along the tube can be determined with high precision. Following centrifugation with a protein mixture, the Proteins distribute into bands along the density gradient. Each band localizes to the region of the tube where the density of the CsCl solution matches the density of the protein. This type of centrifugation boasts exceptionally high resolving power and is particularly successful in separating nucleic acid mixtures.