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

Methods for Experimental Study of Protein Structure
Protein Purification Methods
Assessment of Purification Completeness and Protein Homogeneity

Assessing the purity of an isolated protein requires detecting both protein and non-protein contaminants. The degree of removal of non-protein nitrogenous compounds is usually evaluated by determining the ratio between protein nitrogen (precipitated with 10% TCA) and the total nitrogen content of the preparation.

Evaluating a sample for protein impurities is significantly more challenging. As a rule, a reliable Conclusion can only be reached by combining multiple analytical approaches, most notably ultracentrifugation, Electrophoresis, Chromatography, and molecular (gel) filtration. As experimental techniques advance, the reliability of purity assessments steadily increases. Nevertheless, past and present alike, one can never be entirely certain that an isolated protein sample represents a truly single, homogeneous species. It is not uncommon for a protein deemed homogeneous to turn out heterogeneous when examined by more refined Methods. A case in point is mixtures of Isoenzymes—Proteins with nearly identical properties and the exact same substrate Specificity. For a long time, the existence of these isoforms went completely unnoticed because conventional classical Fractionation Methods failed to separate them.

One must also bear in mind that many proteins can alter their properties during analysis, undergoing Denaturation, for instance. Consequently, this can lead to the erroneous conclusion that two or more distinct proteins are present in the mixture, whereas it actually consists of a single protein in both its native and denatured states.

One of the readily accessible, straightforward, and fairly reliable criteria for protein homogeneity is the solubility test. For a homogeneous protein, this parameter should remain unchanged upon repeating one or more purification steps. Furthermore, the solubility value of an individual protein must be independent of the sample mass taken. In practice, this is easy to test: a series of protein samples with increasing weights is placed into graduated test tubes, and each tube is filled with the same volume of solvent. After complete dissolution, all solutions are filtered, and the protein concentration in each is determined. For a pure individual protein, the plot of The amount of dissolved protein versus the sample mass will consist of two straight segments (see Fig., lines 1 and 2), one of which (line 2) runs parallel to the abscissa axis (representing saturation).

In the presence of protein contaminants, the resulting experimental curve looks different, displaying an intermediate zone between the initial solubility line and the saturation plateau (see Fig.). This extra section (2') appears because the solubility limit of the main protein component in the mixture is reached much sooner than that of the contaminant protein. The increase in Protein solubility (segment 2') continues until the solubility limit of the contaminant protein is also reached. The solubility test stands as one of the most rigorous criteria for protein homogeneity. Conventionally, protein purity is monitored using at least two independent methods. For instance, alongside the solubility test, the purity of an isolated protein is often verified by Analytical ultracentrifugation. The sedimentation pattern of a pure protein should exhibit a single, symmetrical, regularly shaped peak (see Fig.).

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Purity can also be verified using Ion-exchange chromatography, Gel filtration, Thin-Layer Chromatography (TLC), or paper chromatography. In all cases, only a single substance should be detected.