Chromatography affinity, of enzymes

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

The quantitative separation of phosphorus acids and esters by ion-exchange chromatography has been reviewed. The separation of nucleoside polyphosphates by anion-exchange chromatography and the affinity chromatography of enzymes on immobilized adenosine monophosphate have also been described. 

R.R. Maestas, J.R. Prieto, G.D. Kuehn and J.H. Hageman. Polyacryamide-boronate beads saturated with biomolecules A new general support for affinity chromatography of enzymes. 

V. Bouriotis, I.J. Galpin and P.D.G. Dean. Applications of immobilized phenylboronic acids as supports for group-specific ligands in the affinity chromatography of enzymes. J. Chrom. 210 267-278 (1981). 

Affinity chromatography of enzymes from soybean and Takadiastase on glycose-substituted agaroses showed that the p-D-2-acetamido-2-deoxygluco-sidases are not adsorbed specifically, although some purification of these enzymes could be achieved. ... 

Friedberg F, Rhoads AR. Affinity chromatography of enzymes. In Hage DS, editor. Handbook of affinity chromatography 2nd ed. Boca Raton, FL CRC Press 2005. p. 313-46. 

P-Mannanases are generally prepared by conventional chromatographic procedures, which can lead to high yields of enzyme, but in some cases only a poor state of purity. Small quantities of highly purified p-mannanases have been prepared by substrate affinity chromatography of partially purified enzymes on a column of glucomannan immobilised on aminohexane Sepharose 4B (6). The action patterns of enzymes described in this paper were determined using enzymes purified by this latter procedure. 

McCafferty J, Jackson RH, Chiswell DJ, Phage-enzymes expression and affinity chromatography of functional alkaline phosphatase on the surface of bacteriophage, Protein Eng., 4 955-961, 1992. 

Figure 4. Affinity chromatography of diol dehydrase on the adenosyl form of II (37). About 1 unit of enzyme was applied to 1 mL of packed corrinoid gel in 0.1 M potassium phosphate buffer (pH 8.0) containing 2% 1,2-propanediol, in a total volume of 2 mL. Affinity chromatography was carried out as described in the text. Two-milliliter fractions...

The use of enzyme inhibitors is not recommended as they are harmful to human beings. It should be ascertained that the degradation observed is not a function of the analytical assay. Enzyme inhibitors can be used for prevention of enzymatic activity in analytical assays. Mild denaturation may accelerate enzymatic digestion. Selective removal (e.g., affinity chromatography) of specific enzymes should be considered, too. 

Fig. 4.7.2. Affinity chromatography of a-chymotrypsin on inhibitor Sepharose columns. The columns (50x5 mm) were equilibrated and run with O.OS M Tris-hydrochloric acid buffer (pH 8.0). Each sample (2.5 mg) was applied in 0.5 ml of the same buffer. The columns were run at room temperature with a flow-rate about 40 ml/h and fractions containing 1 ml were collected. The arrows indicate a change of elution buffer (0.1 M acetic acid, pH 3.0). (A) Sepharose coupled with e-aminocaproyl-D-tryptophan methyl ester (B) Sepharose coupled with D-tryptophan methyl ester (C) unsubstituted Sepharose. The first peaks in A and B were devoid of enzyme activity. Reproduced with permission from Ref. 46.

Unspecific inhibition of ribonucleotide reduction is produced by compounds like pyridoxal phosphate, or the sulfonated anthraquinone-triazine dye, Cibacron blue. They interact, like in many enzymes, with nucleotide binding domains where pyridoxal phosphate becomes covalently linked to lysine, or in that the dye occupies the whole nucleotide fold. The latter interaction permits its use in affinity chromatography of ribonucleotide reductases Likewise, EDTA is not a specific, nor a potent inhibitor, it may, for example, act by complexation of the structure-stabilizing Mg " ions in native holoenzymes. However the iron-promoted radical regeneration process appears far more susceptible to interference from EDTA ... 

Biocatalysis from discovery to application. Springer, Berlin, Heidelbeig, New York, pp 31-57 Ren X, Yu D, Han S et al. (2007) Thermolysis of recombinant Escherichia coli for recovering a thermostable enzyme. Biochem Eng J 33(l) 94-98 Riechmann L, Kasche V (1985) Peptide synthesis catalyzed by the serine proteinases chymotrypsin and trypsin. Biochim Biophys Acta 830(2) 164-72 Robinson PJ, Wheatley MA, Janson JC et al. (1974) Pilot scale affinity chromatography of p-galactosidase. Biotechnol Bioeng 16 1103-1112... 

Chaga G., Widersten M., Andersson L., Porath J., Danielson U.H. and Maimervik B. 1994. Engineering of a metal coordinating site into human glutathione transferase Ml-1 based on immobilized metal ion affinity chromatography of homologous rat enzymes, Protein Eng., 7, 1115. 

The use of amylose gel cross-linked by epichlorohydrin for affinity chromatography of extracellular isoamylase of Pseudomonas amyloderamosa has been studied. The isoamylase was adsorbed well on the amylose gel and was eluted specifically with maltodextrin. The eluted enzyme was precipitated with ammonium sulphate to remove maltodextrin, and then the solution of the precipitate was dialysed and concentrated by vacuum filtration. By this procedure 96 mg of the enzyme were purified to homogeneity from 20 1 of culture broth in about 70% yield. 

Affinity chromatography of hen egg white lysozyme on chitosan without any intermediate reagent has proved to be a suitable means of purification of the enzyme, subsequently elution being achieved with 1-aminopropane. ... 

Affinity chromatography of light chain iso- 191 enzymes of myosin head region Investigation of binding of fatty acids, 192... 

Affinity chromatography of coenzyme A-dependent enzymes (citrate synthase and succinic thiokinase)... 

Affinity chromatography of soluble membrane 247 receptors from mouse sub-maxiUary glands Affinity chromatographic adsorption of activating factor of lymphocyte adenylate cyclase with consequent reversible deactivation of the enzyme... 

Affinity chromatography of acid ribonuclease 268 from He La cell lysosomes demonstration that the enzyme is a glycoprotein Purification of human liver acid a-D- 148... 

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