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

Isolation and Purification of Proteins
Assessment of Protein Purification Efficiency
Methods for Assessing Protein Purification Efficiency and Homogeneity

The completeness of purification from non-protein nitrogen-containing compounds is usually assessed by The ratio of protein nitrogen (precipitated by 10% TCA) to the total nitrogen of the preparation.

Evaluating the degree of purification from accompanying Proteins presents significant challenges. If the isolated protein exhibits a characteristic biological activity that can be quantified, determining its specific activity—per unit weight of protein or protein nitrogen—is extremely useful. By subsequently calculating the ratio of the specific activity of the obtained preparation to that of the initial tissue homogenate, extract, etc., the so-called purification factor is determined.

The final Assessment of Purification completeness and protein homogeneity requires a combination of several Methods. A preliminary Conclusion regarding homogeneity primarily requires evidence that the preparation cannot be further fractionated by any available methods. Naturally, this refers not to every possible technique, but rather to the most effective variants of principal Fractionation Methods—ultracentrifugation, Electrophoresis, Chromatography, molecular filtration, etc.—applied in analytical rather than preparative modifications. At the same time, one must account for the risk that the detection of new fractions might result from partial Protein Denaturation during some of these Procedures. Conversely, the inability to achieve further fractionation may stem from insufficient resolution of the Separation Methods employed. It has frequently been the case that the application of more advanced techniques enabled the fractionation of proteins previously considered homogeneous.

An important criterion of homogeneity is the solubility test. It is based on the principle that, under identical conditions, the solubility of a pure protein must remain constant regardless of The amount of this protein present as a solid phase. According to the phase rule (Gibbs), the number of degrees of freedom in a system at equilibrium under constant pressure and Temperature is related to the number of components and phases by the following equation:

Class="center">F = C—Р,

where C is the number of components, P is the number of phases, and F is the number of degrees of freedom. Suppose we have a solution of a homogeneous protein. Then the number of components C = 2 (protein + solvent), the number of phases P = 1, and consequently, the number of degrees of freedom F = 1.

This means that both components in such a system can be present in any proportions compatible with their mutual solubility. However, if we consider a saturated solution of the same protein containing a suspended amount of undissolved protein, we are dealing with a two-phase system with zero degrees of freedom. This implies that at any ratio of the phases, their composition must remain invariant. Consequently, no matter how much protein is added to the saturated solution, its concentration will not change, and no additional dissolution will occur.

If increasing amounts of a homogeneous protein are placed in a series of test tubes containing equal volumes of solvent, mixed, allowed to reach equilibrium, filtered, and the protein concentration in the filtrates is determined, the dependence of the latter on the total amount of added protein is represented by the broken line 1 shown in Fig. 6. Segment AB corresponds to the single-phase system, whereas the horizontal portion of the graph represents the two-phase system containing a saturated protein solution of constant concentration, independent of the amount of the solid phase. A different picture is observed when dealing with an unfractionated protein mixture consisting, for example, of two components. In this case, even if the solution is saturated with respect to one of the components, a two-phase system is formed. The concentration of dissolved protein will vary with an increase in the total protein amount, although The rate of these changes will decrease. On the solubility graph of such a protein mixture (curve 2), this state corresponds to the segment B'C'. Thus, the presence of more than one inflection point on the solubility curve, or a generally more complex dependence than that represented by curve 1, serves as evidence of protein heterogeneity. The solubility test is the most rigorous test for homogeneity; however, its applicability is limited by The Need for relatively large amounts of purified protein, the risk of denaturing labile proteins during shaking, and the slow dissolution rate of certain proteins.

Fig. 6. Dependence of dissolved protein concentration on the total protein amount in the system for a homogeneous protein (1) and a mixture of two proteins (2). Explanations are given in the text.

Special mention should be made of assessing protein homogeneity using so-called Immunoelectrophoresis. Its essence lies in the combination of two techniques: 1) separation of a protein mixture by gel electrophoresis; 2) interaction of the resulting fractions with an antiserum containing Antibodies against all Components of the test mixture. The latter is performed in the same gel where electrophoresis was carried out, with the antiserum diffusing in a direction perpendicular to the preceding separation. The co-precipitation zones of each protein with its corresponding antibody generally do not coincide (due to differences in mobility and solubility of the protein–antibody complexes, causing them to travel different distances prior to co-precipitation). In cases where it is possible to obtain an antiserum that precipitates all or most components of the mixture, this method is extremely efficient. It should be kept in mind, however, that obtaining precipitating antisera for many proteins is a formidable task, particularly if their proportion in the mixture is small or if they belong to poorly immunogenic proteins (characterized by a low aromatic amino acid content, relatively small molecular weight, etc.). This significantly restricts the applicability of immunoelectrophoresis.

It has now been proven that the same Cells and Tissues contain extremely closely related proteins that are nevertheless not completely identical in composition, Structure, physicochemical properties, and biological activity. Examples include Isoenzymes and certain proteins resulting from minor alterations in the cellular genome or errors in METABOLISM/35.html">Protein Biosynthesis within the given Cell. This further complicates the evaluation of homogeneity. Even when positive homogeneity data are obtained across all the aforementioned tests, the conclusion should only be considered sufficiently rigorous regarding a very high degree of purification and a high probability that the given protein is homogeneous.