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Review of affinity precipitation methods.


During the 20th century, chromatography, one of the most accurate selective separation techniques and unique in achieving the high standards of product purity is aimed to isolate and purify biological macromolecules and commercial bioproducts (Pecs et al., 1991; Sofer, 1995; Wilkins, 2002). Some chromatography techniques that are using in large-scale include: size-exclusion chromatography, ion-exchange, hydroxyapatite, hydro-phobic interaction chromatography, reversed-phase chromatography, and affinity chromatography (Lyddiatt, 2002). Affinity chromatography, as a powerful tool in biomedical research and biotechnology, was not used in laboratory scale widely because of the high cost of the affinity ligands and their biological and chemical instability (Lowe et al., 2001). However, the new methods has presented for screening, selection and design of stable synthetic ligands recently (Ladner, 2001). Some factors which have efficiency on the success of an affinity chromatographic are: (1) the ligand selectivity, (2) recovery, (3) throughput, (4) reproducibility, (5) stability and maintenance and (6) economy (Turkova, 2002). It is to be notified that ligands for affinity chromatography divided to three groups: protein-structure-based design, protein-function-based design, combinatorial approaches.

During 80s, three techniques are, as novel affinity protein purification methods, which using affinity interacts to provide product with high degree of purity is include: affinity partition, affinity cross-flow filtration, and affinity precipitation (Lowe, 1984). Affinity precipitation is based upon precipitation of the target protein by a smart affinity macroligand (Roy and Gupta, 2002) and several successful purifications of proteins/enzymes by affinity precipitation have been reported (Sharma et al., 2000; Vaidya et al., 2001; Teotia et al., 2001). Here some of these methods and models are explained and reviewed:

Conjugation of PABA-poly (NIPAM)

A mathematical model was proposed to allow the analysis of kinetic enzyme in experimental system. The methodology was tested using a system composed of an enzyme, ethylene glycol and conjugated PABA-poly (NIPAM). The chemical reaction for complex formation and product formation respectively are:



Where, S=substrate, E=enzyme, ES=enzyme-substrate complex, P=product, k1= rate constant for complex formation, k2=rate constant for reverse complex formation, k3=rate constant for product formation. And last formula is:

d{P}/dt = [k.sub.3][E.sub.T]{S}/[k.sub.m](1+{I}/[k.sub.1])+{S} (Eq.3)

Where, P= concentration of product, ES= enzyme-substrate complex, S= concentration of substrate, I= concentration of inhibitor, [E.sub.T]= total complexed and uncomplexed enzyme, k1= rate constant for complex formation, k2= rate constant for reverse complex formation, k3= rate constant for product formation, km= "half-saturation" concentration, k1= "saturation" constant for inhibitor (Syaubari et al., 2008).

Affinity Precipitation by Mosbach and Larsson (1979)

A selective method, called affinity precipitation, was introduced by Mosbach and Larsson (1979) using the dimer of nicotinamide-adenine dinucleotide, oxidized (NAD) for NAD-dependent enzymes such as lactate dehydrogenase (LDH) (See Table 1).

On the other hand, triazine dyes, used as affinity ligands, are known to imitate the coenzyme and thus exhibit a certain affinity towards the cofactor-dependent enzymes such as LDH. Thus, it is logical to use these dyes as specific bifunctional ligands for affinity precipitation. Affinity precipitation, based on a soluble macromolecule (ligand polymer, macroligand), has two functions: (a) it contains an affinity ligand (preferably more, polyvalent macromolecule), (b) it can be precipitated in one of many ways: change in pH, temperature or ionic strength (Pecs et al., 1991). Depend on the nature of macroligands, two ways provided to induce precipitation: homobifunctional ligands and hetero-bifunctional ligands (Hayet & Vijayalakshmi, 1986).

Metal Affinity Precipitation of Proteins

One of methods is metal affinity precipitation of proteins with bischelates of Cu(II) as macroligands. In this method, the known affinity between Cu(II) and metal coordinating residues on the protein surfaces (Van Dam et al., 1989), two cupric cations chelated by molecules of iminodiacetic acid (IDA) immobilized onto a single spacer molecule of polyethylene glycol (PEG) or chelated by a molecule of ethylene glycol bis-aminoethyl crosslink and precipitate proteins that contain multiple metal coordination sites (Vijayalakshmi, 1989).

Metal Affinity Aggregation of Proteins

This method includes modeling the kinetics of the metal affinity aggregation of proteins via a formalism that employs classical gelation theory in conjunction with ion binding equilibrium. Simulations which is using this model,, indicated that the kinetics of coagulation are highly dependent on the pH, pKa, and molar ratio. During the experiment, it has been observed that very high metal ion concentrations and high metal-ligand binding constants serve to reduce the rate of aggregation. High proton concentrations also reduce the rate of aggregation by mass action competition with metal ions for proton binding sites. At the end of experiment, a novel post-gelation solution was applied to account for the finite solubility. This model of the kinetics of metal affinity aggregation would be helpful to find the relation between aggregation process parameters and process performance.

The equations governing the time trajectories of the aggregation reaction were nondimensionalized by the initial concentration of protein in the system, [C.sub.0] or, equivalently, [[mu].sub.0](0); time was nondimensionalized as T= [[mu].sub.0](0)kt. The moments, metal ion concentration, and proton concentration were nondimensionalized as formula (Iyer & Przybycien, 1996):

[[mu].sup.*.sub.n]([tau]) = [[mu].sub.n]([tau])/[[mu].sub.0](0) (Eq.4)

Purification of Lectin by Affinity Precipitation

Lectins from wheat germ, potato and tomato extracts (1.0 ml clarified extracts containing 21 722, 54 650 and 19 280 units) were added to 2 ml of chitosan solution (0.4% wv21 in 50 mM acetate buffer, pH 5.5). The solutions were incubated for different time periods at 258[degrees]C and precipitated by increasing the pH to 7.5 by using 3M NaOH. After centrifugation at 12000 g for 15 min at 108C, the precipitates were washed with 20 mM Tris HCl buffer, pH 7.5, till no activity was detected in the washings. The unbound activity was checked in the supernatant and washings. The bound lectin was eluted by incubating the lectin-chitosan complexes with saturated Mg[Cl.sub.2] for different periods of time at 108C. Mg[Cl.sub.2] at this concentration interfered with lectin determination and was removed by extensive dialysis against respective buffers before determining the recovered lectin. The lectin activity was confirmed by sugar inhibition test using 0.5 M N-acetyl-D-glucosamine (Teotia et al., 2006).

Affinity Chromatography for Purification of Enzymes and Other Proteins

In scale up of affinity chromatography for purification of enzymes and other proteins the following basic assumptions are needed to formulate the rate model: (1) the column is isothermal, (2) the packing particles are considered spherical and possess the same size, (3) the diffusion in the radial direction is negligible, (4) there is no convective flow inside particle macropores, (5) the packing density is even along the column length, and (6) the mass-transfer and kinetic parameters are constant. The second-order kinetics is commonly used to describe the interactions between a macromolecule and an immobilized affinity ligand,

[P.sub.i] + L [??] [P.sub.i]L (Eq.5)

Where [P.sub.i] represents component i (a macromolecule in the mobile phase) and L is the immobilized ligand. The rate equation for Eq. (6) is as follows (Gua et al., 2003):

[partial derivative][C.sup.*.sub.pi]/[partial derivative]t = [][C.sub.pi] ([C.sup.[infinity].sub.i] - [Ns.summation over (j=1)][C.sup.*.sub.pj]) - [k.sub.di][C.sup.*.sub.pi] (Eq.6)

Other method is using k-carrageenan, a naturally occurring family of polysaccharides extracted from red seaweed, for affinity precipitation of pullulanase.

A useful feature of k-carrageenan as a smart polymer is that it becomes insoluble merely upon addition of Kb and no change in pH or temperature is required for reversible solubilization and insolubilization (as is the case of most of the smart polymers, which are pH-responsive or thermo-sensitive) (Mondal et al., 2003).


The Affinity Precipitation Methods which are described in this review are certainly far from being exhausted. The limitation are the ingenuity and skill of biotechnologists. This manuscript is reviewing the affinity chromatography (specifically affinity precipitation) in general that is one of the most suitable research tools for molecular recognition. The new findings have continually caused to change our way of thinking and also to invent new methodology in future.


The financial support by FGRS is gratefully acknowledged. Reference

[1] Gua, T., Hsua, K.-H., & Syub, M.-J. (2003). Scale-up of affinity chromatography for purification of enzymes and other proteins. Enzyme and Microbial Technology 33, 430-437.

[2] Hayet, M., & Vijayalakshmi, M. A. (1986). Affinity Precipitation of Proteins Bis-Dyes. Journal of Chromatography, 376, 157-161.

[3] Iyer, H. V., & Przybycien, T. M. (1996). A Model for Metal Affinity Protein Precipitation. JOURNAL OF COLLOID AND INTERFACE SCIENCE, 177, 391-400.

[4] Ladner, R. C., Ley, A.C. (2001). Novel frameworks as a source of high-affinity ligands. Current Opinion in Biotechnology 12(4), 406-410.

[5] Lowe, C. R. (1984). New developments in downstream processing. Biotechnology, 1(3).

[6] Lowe, C. R., Lowe, A. R., & Gupta, G. (2001). New developments in affinity chromatography with potential application in the production of biopharmaceuticals. Journal of Biochemical and Biophysical Methods 49(1-3), 561-574.

[7] Lyddiatt, A. (2002). Process chromatography: current constraints and future options for the adsorptive recovery of bioproducts Current Opinion in Biotechnology, 13(2), 95-103.

[8] Mondal, K., Roy, I., & Gupta, M. N. (2003). k-Carrageenan as a carrier in affinity precipitation of yeast alcohol dehydrogenase. Protein Expression and Purification, 32, 151-160.

[9] Pecs, M., Eggert, M., & Schiigerl, K. (1991). Affinity precipitation of extracellular microbial enzymes. Journal of Biotechnology, 21(1991), 137-142.

[10] Sofer, G. (1995). Preparative chromatographic separations in pharmaceutical, diagnostic, and biotechnology industries: current and future trends Journal of Chromatography, 707(1), 23-28.

[11] Syaubari, Saari, M., Chuah, T. G., Zulkafli, G., & Ishak, M. (2008). Modeling and kinematic Determination in Affinity Precipitation of Trypsim. Malaysian Journal of Mathematical Science, 2(2), 161-171.

[12] Teotia, S., Mondal, K., & Gupta, M. N. (2006). Integration of Affinity precipitation with partitioning Methods for Bioseparation of Chitin Binding Lectins. Food and Bioproducts Processing, 84, 37-43.

[13] Turkova, J. (2002). Theory and Practice of Biochromatography. London: Taylor and Francis.

[14] Van Dam, M. E., Wuenscheli, G. E., & Arnold, F. H. (1989). Metal affinity precipitation of proteins. Biotechnol. Appl. Biochem., 11, 492-502.

[15] Vijayalakshmi, M. A. (1989). Pseudobiospecific ligand affinity chromatography. Trends Biotechnol., 7(3), 71-76.

[16] Wilkins, M. R. (2002). What do we want from proteomics in the detection and avoidance of adverse drug reactions. Toxicol Lett., 127(1-3), 245-249.

Sa'ari bin Mustapha

Department of Chemical and Environmental Engineering, Faculty of Engineering, University Putra Malaysia, 43400 Serdang, Selangor, Malaysia.

Table 1: affinity precipitation of lactate

 Protein. Activity Yield
 (ma) (I.U.) (%)

Starting solution 2 1105 100
Precipitate 1.8 677 90
Supernatant 0.2 16 10
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Author:Mustapha, Sa'ari bin
Publication:International Journal of Applied Chemistry
Date:Jan 1, 2011
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