Molecular Biology: Protein Structure and Functions - Stepanov V.M. 2005

Primary Structure of Proteins
Proof of Protein Individuality. Protein Microheterogeneity

The Determination of the Introduction/19.html">Primary Structure of a protein is preceded by its purification (see Chapter 3) and the establishment of its chemical individuality, or homogeneity, as it is commonly called. There are no universal criteria for protein homogeneity; the most rigorous proof of its individuality is the unambiguous determination of its primary structure, which requires a great deal of effort and time and is not always feasible. Practically useful, albeit incomplete, criteria of protein individuality include:

✵ the detection of a single protein band upon Disc Electrophoresis or isoelectric focusing, exhibiting the activity characteristic of the given protein; data from Immunoelectrophoresis and immunoblotting are highly conclusive;

✵ the detection of a single band corresponding to the denatured protein upon electrophoresis in a polyacrylamide gel in the presence of an anionic detergent (sodium dodecyl sulfate), especially if immunoblotting confirms that it carries the antigenic determinants of the given protein. In the case of Oligomeric Proteins built from different subunits, there may be several such bands, but the Analysis of the primary structure must be preceded by the Separation of the subunits;

✵ the detection of a single protein peak that is sufficiently symmetrical and coincides with the activity peak across various liquid Chromatography Methods (affinity, ion-exchange, hydrophobic, or Gel chromatography). It is essential that The ratio of activity to protein content (specific activity) remains constant across the entire peak;

✵ the identification of a single amino-terminal sequence. Identifying just the amino-terminal residue for this purpose is rarely used, as the consumption of protein to obtain such limited information is unjustifiably high.

These approaches are neither exhaustive nor mandatory; the proof of individuality is constructed anew for each protein. In doing so, researchers often discover the so-called microheterogeneity of the protein, which is typically caused by ambiguous post-translational modifications or random damage to protein molecules in vivo or during the isolation process. However, heterogeneity resulting from the expression of allelic genes or Alternative Splicing is also possible.

Among the most frequent causes of microheterogeneity are internal cleavages of the polypeptide chain due to the random action of proteinases, so-called frayed ends (the result of ambiguous Processing of the amino-terminal region of the protein), and the deamidation of individual asparagine residues, or more rarely, glutamine. The latter can occur spontaneously, particularly easily within the Asn–Gly sequence, leading to what is known as charge microheterogeneity. Other well-known forms of microheterogeneity include incomplete protein phosphorylation by protein Kinases or differences in The structure of the carbohydrate chains of Glycoproteins.

Occasional suggestions that microheterogeneity might be caused by the existence of stable protein conformers with the same primary structure have not received experimental confirmation and are difficult to explain theoretically. Many types of microheterogeneity do not preclude the determination of the Amino Acid Sequence, but they do complicate the analysis and must be taken into account during its execution.