Enzymes affinity chromatography purification

Milk acid phosphatase has been purified to homogeneity by various forms of chromaotgraphy, including affinity chromatography purification up to 40 000-fold has been claimed. The enzyme shows broad specificity on phosphate esters, including the phosphoseryl residues of casein. It has a molecular mass of about 42 kDa and an isoelectric point of 7.9. Many forms of inorganic phosphate are competitive inhibitors, while fluoride is a powerful non-competitive inhibitor. The enzyme is a glycoprotein and its amino acid composition is known. Milk acid phosphatase shows some similarity to the phosphoprotein phosphatase of spleen but differs from it in a number of characteristics. 

The specific activity of the enzyme was determined in 0.15M lactose solution (0.02M phosphate buffer, pH 7-0). The activity of the enzyme was expressed in terms of units of activity per mg of enzyme. A unit of activity was defined as a p mole of glucose produced per minute. The soluble lactase activity following affinity chromatography purification was 37-1 units/mg. This represented a 4 fold increase in catalytic potency over the specific activity of the crude enzyme preparation (8.9 units/mg). 

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). 

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indicated the presence of small N-glycosylation of all forms of exopolygalacturonases present in carrot roots (unpublished results).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). 

Table 10.3 contains a listing of some important proteins. Protein purification must be done under conditions in which conformational and configurational changes are minimal. Such purification is most often carried out using varieties of chromatography including affinity chromatography and electrophoresis. Some of the common features of enzymes are ... 

Our chromophoric substrates proved to be valuable in the study of several aspects of the enzymology of these cellulases. A rapid and specific method for purification (affinity chromatography) has been developed. Following our collaboration with several groups, new insights into the domain arrangement and tertiary structures of two cellulases were obtained. Contributions to the elucidation of the synergistic action (adsorption-hydrolysis) of these enzymes were achieved. 

FIGURE 13.7 An early example of an enzyme purification performed by affinity chromatography. (Reproduced with permission from Cuatrecasas, P. et ah, Proc. Natl. Acad. Sci. U.S.A., 68, 636, 1968.)... 

Procion Rubine MX-B, Procion Yellow H-A, and Turquoise MX-G. These dyes have found use over the last few decades in the purification of a broad range of proteins and enzymes, including albumin, decarboxylases, glycolytic enzymes, hydrolases, lyases, nucleases, oxidoreductases, synthetases, and transferases [77,78], The first use of dye-ligand affinity chromatography was described by Staal et al. in 1971 [79], Since that time, it has become an extremely popular tool for enzyme and protein purification, with hundreds of such compounds having been isolated by this technique [3-6,76-79],... 

Wikstrom, P. and Larsson, P.-O., Affinity fibre—A new support for rapid enzyme purification by high-performance liquid affinity chromatography, J. Chromatogr., 388, 123-134, 1987. 

The ligands used for enzyme purification can be specific to the desired enzyme (substrate, substrate analogue, enzyme inhibitor, antibody), specific for different classes of enzyme (AMP, NAD, PLP) or of limited predefined specificity (dye affinity chromatography withProcion, Cibacron dyes). 

Although most of the sialidases have been purified by classical methods of enzyme isolation, affinity chromatography is now coming more and more into use, as is indicated by the foregoing examples of purification of sialidases. The adsorbent first used for chromatography of sialidases was sialic acid bound to the surface of intact erythrocytes ... 

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