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

Methods for Experimental Investigation of Protein Structure
Chromatography
Ion-exchange chromatography

The fundamentals of the Ion Exchange principle were established by the Russian botanist, physiologist, and biochemist Mikhail Semyonovich Tsvet. In 1910, he published the THEORETICAL FOUNDATIONS OF the ion exchange method. In 1948, American scientists Moore and Stein developed The Theory of ion-exchange Chromatography as applied to the fractionation of complex amino acid mixtures and introduced it into laboratory practice. This method utilizes ion-exchange polymers (ion-exchange resins). These are typically synthetic polystyrene polymers with chains cross-linked by divinylbenzene, or natural polymers based on Cellulose or Sephadex. Sephadex is a natural dextran polymer, which is a polysaccharide (a glucose polymer) with a molecular weight ranging from 12 million to 1 billion, produced by specific Bacteria. Sephadex is obtained in the form of beads after cross-linking long dextran chains with epichlorohydrin (ECH). Depending on The amount of ECH added (and consequently the number of cross-links), Sephadex gels with varying molecular sieve pore sizes are produced. Depending on the pore size, Proteins of specific molecular weights can penetrate them. For example, Sephadex grades: G-25 up to 400; G-50 up to 10,000; G-75 up to 50,000; G-100 up to 100,000; G-200 up to 200,000.

All the aforementioned polymers, following modification, contain dissociable ionogenic groups on their surface that can exchange ions with corresponding groups on Amino Acids or proteins. Ion-exchange resins are divided into two broad classes: cation exchangers (cationites) and anion exchangers (anionites).

Cation-exchange resins contain the following surface groups:

• -SO3^--M⁺, -РО₃^2- -М⁺, AsO₃^2- -M⁺ - strong cation exchangers.

• -СОО^- -М⁺ - weak cation exchanger.

Anion exchangers:

• -N⁺R₃/A^-, -C₅N⁺/A^- - strong

• -N⁺H₃/A^-, -N⁺H₂R/A^-, -N⁺HR₂/A^- - weak

М⁺ and А^- represent the cation and anion of the resin involved in ion exchange, respectively.

Synthetic ion exchangers are mostly used for the analysis of amino acid mixtures. Fractionating proteins with them is difficult for the following reasons.

1. They have a low binding capacity for proteins. This is due to the high degree of cross-linking in the resin's polymer network, which prevents protein molecules from penetrating into the interior of the ion exchanger, allowing them to interact only with its surface.

2. These resins contain a high density of ionogenic groups (roughly every 10Å). As a result, proteins are held tightly by the resin and are difficult to elute with an eluent. Natural ion exchangers are significantly better suited for proteins, as they have a much lower degree of cross-linking, with ionogenic groups spaced at intervals of ≈50 Å.

Effective ion exchangers for protein analysis include:

> Carboxymethyl cellulose (CM-cellulose). In this cation exchanger, a portion of the cellulose hydroxyl groups are substituted with -ОСН₂СООН groups, which are converted into the salt form -ОСН₂СООNa upon Treatment with alkali. It is a weak cation exchanger that exchanges sodium ions.

> Sulfoethyl cellulose (SE-cellulose) contains the - OCH₂CH₂SO₃H(Na) group and acts as a strong cation exchanger.

> Diethylaminoethyl cellulose (DEAE-cellulose) is one of the most effective anion exchangers for protein analysis. A portion of its hydroxyl groups is replaced by -OCH₂CH₂-N(C₂H₅)₂ groups. These are converted into OH^- or Cl^- forms:

Class="center">[OCH₂CH₂-N⁺H(C₂H₅)₂]•OH^-, [OC₂C₂-N+H(C₂H₅)₂]•Cl^-.

Anion exchangers are more effective for separating acidic and neutral proteins, whereas cation exchangers are better suited for basic proteins.