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

Isolation and Purification of Proteins
General recommendations that are useful to follow during the isolation and purification of proteins

Isolation and Purification of Proteins is one of the most challenging problems in Protein Chemistry. As a rule, biological samples contain mixtures and associations of numerous proteins, many of which share very similar physicochemical properties. It is not uncommon for the specific PHYSICOCHEMICAL CHARACTERISTICS OF the proteins being separated to overlap practically. This necessitates a combination of multiple purification techniques that leverage the widest possible range of differences. The situation is further complicated by the inherent instability of proteins and the risk of Denaturation during purification, which severely limits the application of many effective Methods. All of these factors, combined with the vast Diversity of proteins, make it impossible to recommend a single universal isolation and purification protocol. Even for the exact same protein, several different procedures of comparable efficiency are often proposed.

An important feature of most individual Protein Isolation and purification methods is the Abundance of empirically established procedural details. Strict adherence to recommended protocols—down to seemingly insignificant details (The Significance of which may sometimes remain unclear even to the author of the method)—is a mandatory condition for success.

The following sections outline the fundamental general methods for Protein Purification, along with Examples of how different techniques are combined during the purification of specific proteins. Finally, the criteria for assessing protein purity are discussed.

As a rule, the entire protein isolation process should be conducted at the lowest possible Temperature. Elevated temperatures frequently lead to Protein Denaturation. It is critically important to maintain a low temperature even when a given protein is known to be relatively stable at high temperatures in its natural environment. The reason is that a protein inside a Cell or within crude extracts is often more stable than in its purified form. Isolating proteins at low temperatures also offers the advantage of preventing or minimizing the growth of microorganisms that contaminate non-sterile preparations, while slowing down The activity of hydrolytic Enzymes.

When working with poorly characterized proteins, extreme pH values should be avoided, as the majority of proteins undergo denaturation in strongly acidic or alkaline solutions. Most proteins are most stable near neutral pH, although there are exceptions (for example, Histones, Trypsin, Chymotrypsin, and Pepsin exhibit greater stability at lower pH values).

Many proteins rapidly denature in the presence of agents such as ethanol or acetone. Therefore, special precautions must be taken when using these Solvents—operating at sub-zero temperatures, removing them rapidly, and so forth.

When adding acids, bases, or organic solvents, local concentrations (excesses) must be avoided. It is preferable to add Reagents as dilute solutions, slowly and with continuous stirring. However, when agitating protein solutions, foam formation should be avoided, as this frequently induces protein denaturation at the phase boundary.

During the extraction and purification of proteins, it is highly desirable to remove or inactivate protein-hydrolyzing enzymes (proteases) at the very Cytology/cytology/16.html">Early stages of the Procedure. For instance, when isolating Insulin, the Proteolytic Enzymes of the Pancreas must be inactivated first.

As a rule, the purification processes for most proteins cannot be completed within a single working day. Consequently, there is often a need for short-term storage (up to 1–2 weeks) of intermediate preparations. The optimal storage method is in the frozen state at — 10ч — 20° or lower. However, it should be kept in mind that the freezing process itself leads to an uneven distribution of Water, salts, and protein within the frozen mass, which is not always inert regarding the protein's state. In particular, the freezing of certain Lipoproteins causes their dissociation. For many proteins, short-term storage at 0:+4° is entirely feasible. Concentrated protein solutions and precipitates with a high content of neutral salts (chlorides, sulfates, acetates, phosphates, and carbonates of sodium, potassium, and ammonium) tend to retain their properties best under these conditions.

When long-term storage of intermediate or final purification products is necessary, vacuum freeze-drying—commonly known as lyophilization—is recommended. Currently, there are numerous techniques and specialized devices for lyophilization, the description of which goes beyond The Scope of this manual. It should merely be noted that the residual moisture content after drying must not exceed 3–4% (with ~1–2% being the optimal value). Such preparations can withstand many months or even years of storage at sub-zero temperatures.

In certain cases, protein preparations can be dried using simpler methods. For example, trypsin and chymotrypsin protein precipitates can be dried in a vacuum desiccator over a moisture absorbent (such as calcium chloride). Histones and certain other proteins that tolerate organic solvent Treatment well can be dehydrated with acetone, followed by the removal of the acetone under vacuum.

A specific challenge common to A wide variety of Protein Purification Methods and stages is the Concentration of Protein Solutions. In cases where a mild protein precipitation method is applicable at a given purification step, concentration simply involves precipitation followed by redissolving the pellet in a smaller volume. If precipitation is undesirable for any reason, one must resort to methods varying in complexity. Small volumes of protein solutions are conveniently concentrated by placing them in a cellophane dialysis tubing blown over by air from a fan. Good results are also achieved by dialyzed protein solutions against concentrated solutions or Suspensions of polymers such as polyvinylpyrrolidone, polyethylene glycol, and certain Chromatography resins. Concentration by vacuum evaporation is permissible only under conditions that prevent foaming. If the researcher has access to a sufficiently efficient lyophilization system, the frozen preparation can be vacuum-dried to any desired residual moisture level.

Substantial concentration without denaturation can also be achieved via ultrafiltration.

For highly stable proteins, cruder concentration techniques are sometimes employed (e.g., evaporating solutions at elevated temperatures without a vacuum).