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

Primary structure of the protein molecule
Determination of the number of polypeptide chains. Chain separation

High-molecular-weight Proteins typically consist of multiple polypeptide chains. Generally, each chain has one free a-amino group at the N-terminal end and one a-carboxyl group at the C-terminal end. However, some proteins are an exception to this rule, with their a-amino group being masked by an acetyl or other radical. For instance, in the tobacco mosaic virus (TMV) protein, the N-terminal region of the polypeptide is represented by an N-acetylserine-Tyrosine residue. In such cases, a preliminary deacetylation reaction must be performed before determining the a-NH₂ groups. The number of a-amino groups in a protein molecule, much like the number of a-carboxyl groups, directly indicates the number of polypeptide chains present in the given protein molecule. Thus, the number of polypeptide chains can be determined by identifying the number of N-terminal or C-terminal groups.

As previously mentioned (Chapter III), N-terminal Amino Acids can be determined using Sanger's dinitrofluorobenzene method. Following dinitrophenylation and Hydrolysis of the dinitrophenyl protein derivative (DNP derivative), the reaction products—DNP-amino acids—are adsorbed at pH 7–6 on an anion-exchange resin, whereas free amino acids remain in solution. This occurs because DNP-amino acids exist as anions at this pH, whereas free Amino acids are zwitterions or cations that are not adsorbed by the anion exchanger. The colored derivatives are subsequently eluted from the resin with acid, separated via two-dimensional paper Chromatography, identified using appropriate reference standards, and used to calculate the moles of DNP-amino acids per mole of protein. This allows for the Determination of the number of free N-terminal groups and, consequently, the number of polypeptide chains in the protein. N-terminal amino acids can also be determined as phenylthiohydantoin derivatives or as free acids derived from them using the Edman Degradation method. Finally, C-terminal amino acids can be determined by Hydrazinolysis or the carboxypeptidase method. Determining the number of C-terminal groups is typically employed when the a-amino group is blocked by a radical, as is the case in the TMV protein. In such instances, the number of polypeptide chains must be inferred from The amount of a-carboxyl groups in the protein molecule, which is less reliable (see Chapter III).

Polypeptide chains are linked within a protein molecule through various types of cross-links. Among these, the most important is the bond involving The amino acid cystine, an oxidation product of Cysteine. This amino acid occupies a unique position in Cell/13.html">Protein Structure. As will be shown below (Chapter V), the side chains of cysteine can be linked via oxidation to form cystine, whereby the resulting cystine bridge can "cross-link" both two different polypeptide chains and various segments of a single chain. The presence of such cross-links makes it impossible to dissociate the chains and subsequently study their Amino Acid Sequence. Therefore, it is necessary to disrupt the disulfide bridges and separate the liberated chains. This is best accomplished using a strong oxidizing agent—performic acid—which does not cleave the peptide bond and causes minimal damage to amino acids. In this process, the cystine bridge is oxidized to two molecules of cysteic acid, resulting in the appearance of strongly acidic SO₃H groups on The polypeptide chains:

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The dissociated polypeptide chains can be separated by Ion-exchange chromatography. As mentioned above, it is preferable to use Cellulose-based ion exchangers, which feature high binding capacity for Polypeptides, the absence of irreversible sorption, and other advantages. Using this approach, for example, two Insulin chains and two Hemoglobin subunits have been fractionated and obtained in pure form. In the latter case, the Separation was carried out on carboxymethyl cellulose using gradient elution with a pyridine-formic acid buffer (pH 2.0). The resulting subunits were further separated into individual chains under alkaline conditions.