Allosteric boosting of affinity sorption

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Palladin Institute of Biochemistry of NAS of Ukraine

National University of Food Technologies

SI'O.S.Kolomiychenko Institute of Otolarynglology, NAMSU”

ALLOSTERIC BOOSTING OF AFFINITY SORPTION

Chernyshenko Volodymyr Doctor of Biological Sciences,

Head of the department of protein structure and function

Voronenko Olha Master degree Verevka Serhij Doctor of Biological Sciences, Professor, Head of the Department of Biochemistry

Kyiv

Summary

Affinity chromatography belongs to the classic preparative and research methods of biochemistry, molecular biology, and biotechnology. This method is based on the application of the affinity of biological molecules to complementary structures. One of the complex-forming participants is immobilized on an insoluble matrix, and the second one is in solution. According to a fairly conventional separation, one of these components contains a binding site that interacts with the complementary grouping of the second component. In this work, a complicated variant of affinity sorption is considered, in which the allosteric site additionally participates in the process of complex formation. Using the example of the activation cascade of the blood clotting system, the possibilities of using such a scheme are considered.

Keywords: allosteric site, affinity chromatography, serine proteinases, hemostasis. affinity biological molecule blood

The main text

Various variants of the affinity chromatography method are based on highly selective complexation between biologically active compounds, one of which is covalently immobilized on an insoluble carrier (matrix). This makes it possible to effectively sorb related molecules and separate them from those that do not have similar affinity [1]. According to a fairly conventional separation, one of these components contains a binding site that interacts with the complementary grouping of the second component. Low molecular weight compounds, that correspond to the ligand specificity of the binding site, are often used. As a result of washing the sorbent from ballast substances, the target substance is desorbed using a solution with a free ligand, or using a "deforming buffer" that denatures the sorbed component and converts it into a solution (Fig. 1).

Fig. 1 Classical image of the process of affinity chromatography. A - elution with a soluble counterligand ; B - elution by a “deforming buffer" [1]

Affinity chromatography is used widely among preparative and research methods of biochemistry, molecular biology, and biotechnology [2, 3]. One of the leading parameters of the sorption process is the dissociation constant Kd of the protein complex with the ligand bound by it. The optimal value of this indicator is considered to be 10-5-10^-6 M [4]. On the other hand, interactions with a Kd more than 10^-3 M are not suitable for affinity chromatography, because instead of stable sorption, only deceleration of the protein occurs and its release together with the inactive protein in the form of a blurred peak [5]. However, there are many examples of deviation of the experimental properties of affinity sorbents from the last rule. This is most pronounced for affinity chromatography of serine proteinases. This is due to the presence in the active center of the enzymes of this group not only of the binding site, but also of the allosteric one (S1and S2'-subunits, according to the Schechter-Berger nomenclature [6]. Synchronous interaction of the binding and allosteric sites with the corresponding ligands in terms of specificity ensures a sharp increase in the efficiency of the interaction and has many manifestations both in vivo and in vitro. A thorough review of these examples is given in our previous work, with special attention being paid to the role of the effector site in the waffle sorption of chymotrypsin-like proteinases [7]. It is shown that the synchronous interaction of the binding and allosteric sites of the enzyme with two immobilized hydrophobic ligands can be ensured in two ways - due to a sharp increase in the concentration of the immobilized ligand (Fig. 2, left) or due to immobilization of the ligands through a spacer (Fig. 2, right ). In the first case, there is a classic transition from affinity binding to so-called hydrophobic one [1].

Fig 2 Diagrammatic representation of three different modes of affinity sorption at synchronous interaction by binding site (B) and effectory one (E)

The sorption at high content of immobilized ligand (left) and the interaction of the binding and effectory sites with the pair of ligands that are immobilized via spacer (right). In both cases, instead of the expected weak interaction, exceptionally strong sorption occurs, which can be broken only with the help of a "deforming buffer" [8]. A similar phenomenon can be defined as an allosteric boosting of affinity sorption. It is not limited to proteolytic enzymes alone and is observed for a variety of proteins exhibiting binding properties. A striking example of such bi-molecular binding is the interaction of p-lactate dehydrogenase with immobilized adenosine-5'monophosphate [9,10]. In the case of this enzyme, as the concentration of the immobilized ligand increases, a jump-like transition from unbinding to binding is observed. The concentration dependence of the nature of the interaction of this enzyme with the affinity sorbent on the concentration of the immobilized ligand has a pronounced sigmoidal character, which indicates the statistical nature of the distribution of distances between the molecules of the immobilized ligand exposed for interaction with the enzyme. Upon reaching a certain threshold value, the number of available ligand molecules located at a distance necessary for effective interaction with both the binding sites and effector one of the enzyme becomes statistically significant and begins to make an appropriate contribution to the nature of the sorption process. A further increase in the concentration of the immobilized ligand leads to a sharp increase in the number of such pairs with a corresponding change in the nature of binding. Saturation occurs when the concentration of the immobilized ligand reaches a value that ensures the presence of at least one optimally positioned ligand pair on the area occupied by one enzyme molecule on the surface of the matrix available for contact. A further increase in the concentration of the immobilized ligand is insignificant for either quantitative or qualitative characteristics of the sorption process. Analogous phenomena are inherent in the interaction of y-globulins with sorbents containing hydrophobic ligands. Similar to the above examples, desorption is achieved only when using a "deforming eluent" [11 ].

The allosteric enhancement of sorption according to the scheme of Fig. 2 (right) deserves no less attention. According to the classic views of Cuatrecasas P., the role of the spacer is to increase the steric accessibility of the immobilized ligand [12]. However, no spacer can provide greater steric accessibility to the ligand than during the last stay in the free dissolved state. In this regard, it is worth considering typical examples of affinity sorption of trypsin-like proteinases on sorbents with immobilized low-molecular-weight ligands. As we;; known, unlike chymotrypsin-like proteinases, the specificity of the binding site of trypsin-like ones does not correspond to hydrophobic, but to positively charged amino acid residues. When immobilized by the a-amino group, neither lysine nor arginine provides trypsin sorption [13,14]. Instead, immobilization through a spacer turns the C-terminal arginine into a highly effective ligand, which ensures effective binding of trypsin to the appropriate sorbent [15-17]. That is, sorption occurred at the expense of both the binding site and the allosteric site according to the scheme of Fig. 2, left.

Among the synthetic compounds - ligands of affinity sorbents for trypsin-like proteinases - a separate and rather large group consists of benzamidine derivatives - a competitive inhibitor of trypsin that effectively interacts with the binding site of the enzyme (Fig. 3).

Fig. 3 Benzamidine (left) and para-amino-benzamidine (right)

When para-amino-benzamidine is immobilized directly on the activated matrix, a sorbent is formed that is unable to effectively bind trypsin-like enzymes. Immobilization of this ligand through a spacer - 6-aminohexanoic acid or 1,6diaminohexane - ensures extremely strong binding, which requires desorption with "deforming eluents" [18-21 ].

The last circumstance from the point of view of pharmaceutical biotechnology is extremely undesirable. During any renaturation processes, an admixture of irreversibly denatured protein remains in the final product, which can cause the development of autoimmune complications [22]. The question arises about the possibility of creating affinity sorbents that effectively bind the target protein and allow it to be desorbed under mild conditions. In our opinion, there are necessary prerequisites for this.

In all the considered examples, the ligands of both the binding site and the allosteric site are the same compounds. However, for the areas of activation cleavage of proenzymes of the blood coagulation system and inhibitors of the corresponding enzymes, a pronounced dominance of hydrophobic amino acids is observed in the P2^Lpositions of the cleavable bonds complementary to the effectore sites [23]. P1positions complementary to the binding sites are occupied exclusively by arginine residues, as it should be for trypsin-like enzymes. This creates prerequisites for allosteric enhancement of affinity sorption of the corresponding proteins according to the scheme shown in Fig. 4.

Fig. 4 Allosteric boosting of affinity sorption by an excess of a soluble ligand complementary to the effector site

The use of an eluent that does not contain ligands of the allosteric site makes it possible to achieve soft desorption of the target protein without the development of denaturation processes. Countless examples of changes in the associative properties of enzymes under allosteric activation testify to the possibil ity of such a scheme. In particular, during the hydrolysis of N-benzoyl-L-arginine ethyl ester by thrombin, allosteric activation by isopropanol leads not only to an increase in kcat, but also to a decrease in Km [24]. It is also worth paying attention to the question of the possibility of using allosteric boosting for isolation both active enzymes and proenzymes. Some evidence in favor of this possibility was obtained in the study of sorption of trypsin and trypsinogen on a sorbent with immobilized trisilol [25].

Summarizing the above, we can conclude that the use of allosteric amplification s expands ignificantly the possibilities of affinity chromatography.

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