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

Isolation and Purification of Proteins
Extraction of Proteins from Cells and Tissues

Typically, at this stage, the protein is not only extracted but also subjected to preliminary purification.

If the protein is localized in the extracellular fluid, mechanical expression of the tissue "juice" is sometimes sufficient. However, high yields of intracellular Proteins can only be achieved after tissue disruption. In many cases, grinding in a mortar or using conventional meat grinders (including the Latapi mill, which ensures finer comminution of small tissue samples) is sufficient. Nevertheless, homogenization using specialized devices is more efficient, convenient, and reproducible. These include blenders, where pieces of tissue or coarse minces suspended in liquid are disrupted by high-speed rotating blades, and homogenizers, where disintegration occurs through the grinding of tissue between Glass or plastic surfaces (with an adjustable gap depending on the desired degree of disruption). Homogenizers of the first type have a higher throughput, but exert a relatively harsh effect on the Cells. The second type allows for precise control over the degree of homogenization, making it possible to isolate intact subcellular structures such as nuclei.

Often, complete Cell lysis requires considerable effort. In addition to mechanical disruption, cycles of rapid freezing and thawing can be useful here. Good results are frequently obtained by rapid freezing in liquid air or nitrogen, followed by grinding the frozen sample in a mortar or porcelain mill. For particularly resilient cells (such as certain Bacteria) and for the deep disruption of specific animal cell Organelles (e.g., Mitochondria), ultrasonic Treatment is recommended. By adjusting the power output and frequency, it is generally possible to select conditions that avoid Denaturation of the target protein.

Many relatively stable Proteins can be successfully extracted from tissue preparations after treatment with agents such as acetone or glycerol solutions. Finally, extraction is facilitated if the tissue samples have been pre-lyophilized.

The COMPOSITION OF THE extractant is usually chosen to ensure the maximum yield of the target protein while leaving behind (or even coagulating) impurities. For instance, extracting proteinases from the Pancreas with 0.25 N sulfuric acid or extracting Histones from cell nuclei with an aqueous Hydrochloric acid-ethanol mixture yields good results. However, such harsh extractants are unsuitable for less stable proteins, which require a more delicate optimization of pH, Ionic strength, Temperature, extraction time, and solvent composition.

It should be emphasized that when varying the concentration and ratio of different salts, one must focus not only on molar concentration, but primarily on the ionic strength of the solution.

The ionic strength I (or μ) is equal to half the sum of the products of the concentration C of each ion present in the solution and the square of its valence (charge) Z:

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where the index i is the sequence number assigned to the ion in the given solution containing a total of n ions.

By utilizing the ionic strength of the solution, one can best account for the combined effect of the concentrations and charges of all ions.

A clear example of a complex Protein Extraction regime is the multi-stage isolation of Myosin from Muscle tissue.

The lability of this protein requires all operations to be performed at 0–4°C and a pH exceeding 6.5. Working at such temperatures is also necessary to maintain a certain amount of adenosine triphosphate in the muscle and muscle homogenate, which promotes the dissociation of the main accompanying protein, Actin, and increases myosin extractability. The choice of ionic strength is dictated by the fact that at μ = 0.3, myosin extraction proceeds very slowly, whereas increasing the ionic strength rapidly raises the proportion of co-extracted actin. Typically, an ionic strength close to 0.5 is used. Similar complications arise when choosing the optimal pH value. Rapid and complete extraction is achieved at pH 7.5–8.0; however, this results in significant actin contamination. Therefore, one usually accepts the loss of a fraction of the myosin for the sake of higher purity at this stage and employs pH values in the range of 6.7–7.0. Of no less importance is the duration of extraction, which is generally limited to 15–30 min, again to reduce actin contamination.

A particularly challenging task is the isolation of poorly soluble proteins, such as Scleroproteins (proteins of integumentary and Connective Tissues, including keratin, Silk Fibroin, etc.). In such cases, after removing the bulk of soluble proteins, researchers attempt to exploit the resistance of scleroproteins to the action of Pepsin or Trypsin, using these Enzymes to digest the remaining accompanying proteins. Frequently, in order to study specific compositional and Structural Features of these proteins, researchers are forced to deliberately dissolve them under conditions that cause partial protein fragmentation (in solutions of alkalis, urea, lithium bromide, reducing agents that cleave Disulfide Bonds, etc.).