Practical Protein Chemistry - A. Darbre 1989

Determination of the composition of protein oligomers. Preparation of monomers and polypeptide chains
Stoichiometric ratio of monomers in the oligomer
Hybridization

Information regarding the subunit composition of oligomers can be obtained through in vitro hybridization of two distinct purified protein forms. These may be homologous Proteins, Isoenzymes, or chemically modified oligomers; the only crucial requirement is that the two protein forms possess distinctive features that allow the resulting hybrid molecules to be differentiated and quantified. The number of monomers within an oligomer can be determined from the quantity of hybrids formed. For instance, assuming a tetramer assembles from identical subunits α and β, five different complexes may arise: αααα, αααβ, ααββ, αβββ, and ββββ.

If the monomers are not identical (specifically, aspartate transcarbamylase contains 6 catalytic and 6 Regulatory Subunits), the task becomes considerably more complex. When the subunits are different yet structurally and functionally equivalent, the number of possible hybrids, $N$, is determined by the following formula:

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where $s$ is the number of subunits, and $m$ is the total number of different unit types.

Researchers often lack the homologous proteins (or isoenzymes) required for hybridization; therefore, chemical modification is recommended in the general case. In investigating the subunit Structure of aldolase, modification was carried out using succinic anhydride [120]. As a result, positively charged Lysine residues acquired a negative charge. Anhydrides of other dicarboxylic acids can also be employed, although the reaction with them is reversible. This method of subunit modification may lead to oligomer dissociation due to electrostatic repulsion between similarly charged groups. The dissociation issue can be resolved by partial acylation with succinic anhydride, though this introduces The Challenge of obtaining homogeneous preparations. The hybridization method yields no results if the protein exhibits a different quaternary structure before and after modification, or if it fails to reconstitute altogether following dissociation into monomers.

1.4.3.1. Procedures. Below are two exemplary procedures for assembling hybrid proteins from homologs and chemically modified subunits.

Hemoglobin hybridization [178]. A 1:1 mixture of hemoglobin S and radiolabeled hemoglobin A is dialyzed against 0.1 M sodium acetate buffer (pH 5) at 3 °C for 48 h to dissociate the protein into α- and β-chains. Assembly is carried out by subsequent dialysis against distilled Water for 24 h. Radiolabeled hemoglobin S—a hybrid protein formed via the exchange of α-chains between hemoglobin A and S—is isolated chromatographically.

Aldolase hybridization [120]. Native and succinylated aldolases are mixed in various molar ratios (protein concentration 0.4%) in 0.02 M potassium phosphate buffer (pH 6.5) containing 4.0 M urea, 0.002 M EDTA, and 0.1 M dithiothreitol at 4 °C. After 30 min, the samples are dialyzed for 12 h to remove urea against a buffer containing 0.5 M NaCl and 0.2 mM dithiothreitol, and then dialyzed 2–3 times against a buffer devoid of NaCl.