Enzymes, affinity chromatography

Sepharose (e.g. Sepharose CL and Bio-Gel A) is a bead form of agarose gel which is useful for the fractionation of high molecular weight substances, for molecular weight determinations of large molecules (molecular weight > 5000), and for the immobilisation of enzymes, antibodies, hormones and receptors usually for affinity chromatography applications. 

EC 3.4.24.3]. Purified by using /V-ethylmaleimide to activate the enzyme, and wheat germ agglutinin-agarose affinity chromatography [Callaway et al. Biochemistry 25 4757 1986],... 

Reverse transcriptase (from avian or murine RNA tumour viruses) [9068-38-6] [EC 2.7.7.49]. Purified by solubilising the virus with non-ionic detergent. Lysed virions were adsorbed on DEAE-cellulose or DEAE-Sephadex columns and the enzyme eluted with a salt gradient, then chromatographed on a phosphocellulose column and enzyme activity eluted in a salt gradient. Purified from other viral proteins by affinity chromatography on a pyran-Sepharose column. [Verna Biochim Biophys Acta 473 1 7977 Smith Methods Enzymol 65 560 1980 see commercial catalogues for other transcriptases.]... 

The fact that adenosine and its derivatives are azo coupling components is used for immobilizing nicotinamide-adenine nucleotide (NAD+) for affinity chromatography purposes. In 12.58 NAD+ is bonded to a matrix through an azo bond. Compound 12.58 is used for the purification of dehydrogenase enzymes (Hocking and Harris, 1973). 

Affinity chromatography exploits the high selectivity of most proteins for their ligands. Enzymes may be puri-... 

Recombinant fusion proteins such as His-tagged or GST fusion enzymes are readily purified by affinity chromatography. 

Affinity chromatography (12) has become an important tool in the isolation of purified fractions of such substances as enzymes. Advantage is taken of specific interactions such as antigen-antibody interactions. One substance of the pair (e.g. antigen) is bonded to a support. When a mixture is passed through the column, the specific interaction retains the corresponding antibody relative to other substances. A change of mobile phase conditions then elutes the pure antibody. This method has a real potential for analysis of specific proteins in body fluids. 

The affinity chromatography on ConA - cellulose indicated the presence of small N-glycosylation of all forms of exopolygalacturonases present in carrot roots (unpublished results). This method was usefull for purification of these enzymes from other protein inpurities but was completely uneffective by separation of individual forms (Fig. 4). 

The clearing effect of heparin on blood is associated with release of the triglyceride-hydrolyzing enzyme lipoproteinlipase (LPL) from the surface of endothelial cells.458,459 In addition to a number of apparently equivalent lipases from different tissues,459 heparin also releases a hepatic lipase.460,461 As suggested by the results of affinity-chromatography studies, this release is probably associated with binding of the polysaccharide to the enzyme. 62,463 Because other polyanions,464 including the... 

One limitation to this method should be noted. If the antibody-enzyme conjugate is prepared using antibody fragments such as Fab or F(ab )2, then nickel-chelate affinity chromatography will not work, since the requisite Fc portion of the antibody necessary for complexing with the metal is not present. 

Figure 20.15 An affinity chromatography support containing iminodiacetic acid groups chelated with nickel may be used to remove excess enzyme after reactions to produce antibody-enzyme conjugates. The nickel chelate binds to the antibody in the Fc region, retaining the conjugate while allowing free enzyme to pass through the gel unretarded.

Lowe, C.R., and Dean, P.D.G. (1971) Affinity chromatography of enzymes on insolubilized cofactors. FEBS Lett. 14, 313-316. 

Compared with enzymes fewer reports are available on immobilization of antibody (Ab) in sol-gels and their applications in immunosensing. Immobilization of Abs on a solid support was first reported in 1967 [128] and the technology has widespread application in affinity chromatography and other areas. However, the major problem associated with covalent immobilization of antibody on solid surface is partial loss of biological activity due to the random orientation of the asymmetric macromolecules,... 

Although affinity chromatography has not been used directly as an analytical method, it may be modified in the future to produce a viable technique. Leucovorin has been used as an effective spacer in obtaining active samples of dihydrofolate reductase.79 If the enzyme could be immobilized without losing its activity, perhaps it could be used to separate folates. 

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