Purification of normal human urinary N-acetyl-beta-hexosaminidase A by affinity chromatography.

N-Acetyl-beta-hexosaminidase A was purified 1000-fold from human urine by chromatography on DEAE-Sephadex followed by concanavalin A--Sepharose affinity chromatography. The optimal pH range was 4.4--4.5 for both the N-acetylglucosamine and N-acetylgalactosamine derivatives. The Km values were 0.51 mM and 0.28 mM respectively for the N-acetylglucosamine and N-acetylgalactosamine derivatives. The glycoprotein nature of the urinary enzyme was established by its affinity towards concanavalin A as well as by the presence of sialic acid, galactose, glucose, mannose and hexosamines in the molecule.     

Purification of phosphotransacetylase by affinity chromatography.

Phosphotransacetylase from Clostridium kluyveri was purified using a C⁸-(6-aminohexyl)-amino-desulfo-coenzyme A-Sepharose column. The method of synthesis of the affinity matrix is described. A crude extract was treated with ammonium sulfate and chromatographed on the desulfo-coenzyme A-Sepharose column. Using this method the enzyme was purified 83-fold and was found to be 73% pure. A new method for the determination of the purity of phosphotransacetylase by activity staining of polyacrylamide gels with 5,5′-dithiobis(2-nitrobenzoic acid) is described.     

Purification of human liver glycoprotein sialyltransferase by affinity chromatography

A simple procedure has been developed for purifying solubilized human liver glycoprotein sialyltransferase (EC 2.4.99.1) 16 000-fold in 4–5% yield. The procedure involves two centrifugation steps, affinity chromatography of the ultrasupernatant fluid on cytidine diphosphate-hexanolamine-agarose followed by gel filtration on Sephadex G-150. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) indicated that the purified sialyltransferase preparation contains approximately equivalent amounts of three protein bands (with apparent molecular weights of 61 000, 63 000 and 70 000) and is highly purified if not homogeneous.     

Purification of streptokinase by affinity chromatography on immobilized acylated human plasminogen.

Streptokinase is an extracellular protein produced by several strains of streptococci. It functions in the species-specific conversion of plasminogen to plasmin. In this paper we describe the purification of streptokinase by affinity chromatography on human plasminogen acylated with p'-nitrophenyl p-guanidinobenzoate. The acylated and non-acylated plasminogen and plasmin were coupled to cyanogen bromide-activated Sepharose 4B and evaluated for streptokinase purification. These results show that a homogeneous preparation of streptokinase with high specific activity and high yield can be obtained using acylated plasminogen. This method permits the binding of one milligram of streptokinase per milliliter of swollen gel.     

Purification of porcine enterokinase by affinity chromatography.

A method is described for the purification of porcine enterokinase by affinity chromatography with p-aminobenzamidine as the ligand. Purification was completed by immunoadsorption with antisera raised to components binding non-biologically to the gel. The final enterokinase preparation was 2.3 times more active than the most active preparation previously described.     

Purification of acetate kinase by affinity chromatography.

A one-step procedure using affinity chromatography has been shown to purify to apparent homogeneity acetate kinase from a commercially available preparation and to partially purify the enzyme from a crude, cell-free extract. Since the gel's capacity for enzyme adsorption is controlled by the thermodynamics of ligand-enzyme interaction, maximization of the adsorption isotherm was attempted. Enzyme adsorption decreased logarithmically with increasing ionic strength but increased with increasing concentration of MgCl2. These competing effects caused the net adsorption of enzyme to increase to a maximum and then to decrease as the MgCl2 concentration was raised. The results allow a significant improvement in affinity column performance and have important implications for scale-up procedures.     

Purification of human immunoglobulin M by affinity chromatography on protamine-sepharose

Human IgM has been isolated from plasma by a simple procedure in high yield. The first step was adsorption to protamine-Sepharose and elution by increasing the ionic strength with NaCl. This was followed by two gel filtration steps resulting in a 98% pure IgM in about 30% yield. A somewhat modified procedure could also be used for purification of IgM from Cohn fraction II + III. The purified IgM was found to have a sedimentation constant in agreement with reported values. In immunoelectrophoresis and isoelectric focusing, purified IgM showed the same behaviour as IgM in plasma. Different fragments of IgM were tested for binding to the protamine-Sepharose adsorbent. IgM_S and Fab were not bound unlike (Fc)₅, indicating that the sites responsible for binding are located in the Fc part and that several Fc parts are necessary for sufficiently strong binding for adsorption.     

Purification of human serum angiotensin I-converting enzyme by affinity chromatography

A relatively simple procedure is described for purifying human serum angiotensin-converting enzyme. The enzyme was purified 130,000-fold to electrophoretic homogeneity using affinity chromatography as the principal purification step. The ligand was an immobilized competitive inhibitor, d-cysteinyl-l-proline. A six-carbon spacer arm was satisfactory for trapping the enzyme; 80% of the bound enzyme was eluted with 3 m urea-1.0 m NaCl-0.1 m Tris, pH 8.3. The specific activity was 39 units/mg protein and the molecular weight (155,000), isoelectric point (4.7), kinetic properties, and the effect of various inhibitors are in agreement with published reports.     

Purification of ficin by affinity chromatography

The sulfhydryl proteinase ficin (EC 3.4.4.12) was purified by chromatography on an agarose-mercurial column. Two separate protein fractions were eluted, ficin and mercurificin, both exhibiting enzymatic activity upon activation by excess thiol.     

Purification of antibodies by affinity chromatography

This review focusses on affinity purification of immunoglobulins, a methodology which is a powerful tool to obtain pure and intact antibodies. Affinity techniques allow antibody purification both in a single step chromatographic procedure as well as in complex purification protocols depending on the intention to use the target antibody. The purification strategies for antibodies by interaction with affinity ligands such as antibodies and Fe receptors or low molecular weight compounds are described.     

Purification of alternanase by affinity chromatography

The enzyme alternanase, produced by Bacillus sp. NRRL B-21195, hydrolyzes alternan, a polysaccharide produced by certain strains of Leuconostoc mesenteroides that consists of glucose linked by alternating α(1→6), α(1→3) linkages. The main product of enzymatic hydrolysis by alternanase is a novel cyclic tetrasaccharide of glucose that also has alternating linkages between the glucose moieties. An improved purification scheme for alternanase has been developed that incorporates the use of isomaltosyl units linked to agarose for selectively binding the alternanase enzyme. Bound enzyme was eluted with 0.5 M sodium chloride and was nearly pure after this procedure. When followed by preparative isoelectric focusing, a single band of 117 kDa was measured when the purified protein was analyzed by HPLC size-exclusion chromatography/multiangle light scattering. The purification procedure can be scaled to permit large quantities of enzyme to be purified in high (36%) yield. Electronic Publication