Purification and immobilization of rabbit liver aldehyde oxidase

Aldehyde oxidase (E.C. 1.2.3.1) was isolated from rabbit liver and two potential bioaffinity ligands, i.e., 3-aminocarbonyl-1-benzyl-6-methylpyridinium bromide and 3-aminocarbonyl-1-benzyl-4,6-dimethylpyridinium chloride, were tested for their applicability in a purification procedure for this enzyme. Various supports and different coupling methods were investigated for the immobilization of aldehyde oxidase. Adsorption to n-hexyl- and n-octylamine-substituted Sepharose 4B and DEAE Sepharose 6B gave the best retention of aldehyde oxidase activity. The storage stability of free enzyme and enzyme immobilized to n-octylamine-substituted Sepharose 4B was studied in several buffers at pH 7.8 and 9.0. This showed that the stability of immobilized enzyme was much less than that of free enzyme. The apparent operational stability of the immobilized enzyme preparation, however, improved substantially compared to soluble enzyme, although the corresponding product yield is still very poor. Coimmobilization of catalase and/or superoxide dismutase provided no significant increase of the apparent operational stability and product yield. A positive effect on both parameters was found for aldehyde oxidase-n-alkylamine Sepharose 4B preparations by increasing the amount of enzyme adsorbed per unit weight of support, whereas the productivity of these preparations remained about constant.     

Purification of human milk bile salt-activated lipase by cholic acid-coupled sepharose 4B affinity chromatography

Sequential chromatography of human milk whey on concanavalin A—Sepharose 4B followed by cholate—Sepharose 4B yielded a bile salt-activated lipase with 150-fold purification. The lipase was not retained by concanavalin A—Sepharose 4B but was retained by the cholate—Sepharose 4B, from which it was eluted with 2% sodium cholate. The affinity chromatography procedure on cholate—Sepharose 4B was based on the specific structural requirement of the enzyme for a 7-hydroxyl group of bile salt. Sodium deoxycholate, which lacks the 7-hydroxyl group, was effective in removing the nonspecifically bound proteins without affecting the binding of the enzyme. Bile salt-activated lipase showed a single band on urea-sodium dodecyl sulfate—polyacrylamide gel electrophoresis with an apparent molecular weight of 125,000, and based on densitometric measurement accounted for 0.5–1.0% of the protein mass of human whole milk. A rabbit antiserum to the purified bile salt-activated lipase caused no inhibition of human milk lipoprotein lipase activity but completely inhibited bile salt-activated lipase activity.     

Identification of plasmin as the major contaminant in immunoglobulin preparations

A proteolytic enzyme could be isolated from rabbit serum by means of DEAE cellulose, Protein A-bound Sepharose and lysine-bound Sepharose chromatographies. This enzyme was found to be the major protease contaminating IgG preparations of rabbit serum. This enzyme was identified as plasmin because it displayed an apparent Mr of 90,000 on nonreduced SDS polyacrylamide gel electrophoresis, was able to directly lyse fibrin and the chromogenic substrate H-D-Val-Leu-Lys-p-nitroanilide, and was stable after heating at 56 degrees for 30 min but broke down at 80 degrees. Its Km toward the chromogenic substrate was 0.35 mM, which agreed well with the published value for plasmin.     

Purification of the gonadotropin-releasing hormone-degrading enzyme by affinity chromatography.

A crude preparation of Kallikrein inactivator, which inhibits the gonadotropin-releasing hormone (GnRH)-degrading enzyme(s) from rat hypothalamus and anterior pituitary, was fractionated by passage through an ion-exchange column. The enzyme-inhibiting fraction was coupled to Sepharose and the resin obtained was used for, affinity-chromatography purification of the GnRH-degrading enzyme. The enzyme from crude tissue preparations was retained on this column and eluted by 0.05 M phosphate buffer. A 9-12 fold increase in the specific activity of the enzyme was achieved. Bacitracin, an effective peptide inhibitor of the degradation of GnRH, was also coupled to Sepharose. Three different such Sepharose-bacitracin conjugates were synthesized, two of which inhibited the degradation of GnRH by hypothalamic and pituitary extracts. They all failed, however, to separate the active enzymic fraction from the bulk of accompanying proteins, using affinity chromatographic techniques.     

Covalent immobilization of cyclodextrin glucosyltransferase (CGTase) in activated silica and Sepharose

Cyclodextrin glucanotransferase is a non-Leloir glycosyltransferase that directly employs the free energy of cleavage of starch to produce cyclodextrins. In presence of appropriate acceptors, this enzyme synthesizes oligosaccharides containing alpha(1-->4) bonds. We have investigated the covalent immobilization of CGTase onto different activated supports. Silica was aminated and further activated with glutaraldehyde. The maximum amount of bound protein was about 4 mg CGTase per gram of support; however, the catalytic efficiency of the immobilized enzyme was lower than 6%. Sepharose 4B activated with cyanogen bromide (CNBr-activated Sepharose) and Sepharose 4B with a spacer arm of 1,6-diaminohexane (EAH Sepharose) were also assayed. These gels react with the amino and carboxylic groups of CGTase, respectively. With CNBr-activated Sepharose, a low percentage of enzyme was bound to the support but with a significant catalytic efficiency (29%). A higher recovery of protein was obtained with EAH Sepharose (62%), but only 2.4% of the initial activity was present in the immobilized biocatalyst. The results were discussed in terms of CGTase structure and mechanism. In addition, the solvent accessibility of amino or carboxylic groups, calculated using the NACCESS software, was considered.     

Method of purification of glucose oxidase by means of affinity chromatography on immunoabsorbent]

The possibility to purify glucose oxidase from Penicillium vitale on immunosorbent containing specific antibodies to the enzyme covalently bound with Sepharose 4B is studied. The method of affinity chromatography was applied, beside routine methods of fractionating blood serum proteins, to isolate specific antibodies from antiserum of rabbits immunized with glucose oxidase. Immobilized on Sepharose glucose oxidase was used as biospecific sorbent. Specific antibodies to the enzyme were isolated using chromatograpy of gamma-globulins mixture followed by protein desorption from the column with 1 M NaC1 and 3% glucose. Antibodies were immobilized by their covalent binding to activated Sepharose. The immunosorbent obtained was used to purify low active preparation of glucose oxidase by means of affinity chromatography under conditions worked out for the antibodies isolation. The enzyme was eluted from the column with 1 M NaC1 (pH 3.0) containing 3% glucose. 5-Fold purified enzyme preparation was isolated.     

Alteration of processive mechanism of native polynucleotide phosphorylase by fixation to solid matrix.

Native $\text{E. coli}$ polynucleotide phosphorylase can be covalently bound to BrCN activated Sepharose. The Sepharose bound enzyme retains 70 % of its initial activity in polymerisation of nucleoside diphosphate. The Km of the enzyme for the polymerisation reaction in comparison to the soluble enzyme is not affected by its linkage to a solid matrix. The phosphorolysis of an hexanucleotide by the Sepharose-bound enzyme is not affected either. However, the rate of phosphorolysis of a long chain polynucleotide is dramatically altered. The Km values for poly(A) or poly(U) are increased by two orders of magnitude. The decrease of affinity for polymeric substrate is accompanied by a significant modification of the known processive mechanism characteristic of the native soluble enzyme.     

Affinity chromatography and separation of the molecular forms of monkey brain alpha-L-fucosidase on fucose-linked sepharose.

A simple affinity system which required coupling of alpha-L-fucose to Sepharose 4B by epichlorohydrin treatment of Sepharose 4B in the presence of alpha-L-fucose under alkaline conditions has been described. A partially purified preparation of monkey brain alpha-L-fucosidase (alpha-L-fucoside fucohydrolase, EC 3.2.1.51) was resolved at pH 5.0 into two major fractions: one bound and one retarded. The enzyme bound to the affinity column and specifically eluted by 2 mM alpha-L-fucose at pH 5.0 appeared to be homogeneous by polyacrylamide gel electrophoresis and was constituted mainly by the tetrameric form of the enzyme. The enzyme fraction retarded by the affinity column was found to contain mainly the monomeric form of the enzyme. Additional evidence for the different molecular forms of the enzyme in the bound and retarded fractions came from pH activity profiles and heat inactivation studies. The fucose-Sepharose appeared to bind the tetrameric form of the enzyme specifically and, further, alpha-L-fucose helped to retain the molecular integrity of the tetrameric enzyme.     

Isolation of neutral proteinase of ribosomes (cathepsin R)

A method for isolation of cathepsin R from rat liver ribosomes allowing for a 264-fold increase of specific activity is described. The purification procedure includes enzyme extraction from ribosomes with 2-4 M LiCl and two-step affinity chromatography on Sepharose with immobilized soy bean trypsin inhibitor and trypsin-Sepharose.     

A simple procedure for isolating adenosine triphosphatase from mitochondria.

A simple method for isolation of adenosine triphosphatase (EC 3.6.1.3) from mitochondria is described. The enzyme is released from mitochondrial Lubrol particles by drastic sonication and purified by gel filtration on Sepharose 6-B. The described procedure is effective in isolating adenosine triphosphatase from rat liver as it is from beef heart mitochondria. The enzyme isolated from beef heart has a specific activity of 120 mumol P/min per mg protein and enzyme isolated from rat liver has a specific activity of 70 mumol P/min per mg protein when measured as a release of inorganic phosphate.     

Bioaffinity Based Oriented Immobilization of Stem Bromelain

Bromelain is a basic, 23.8 kDa thiol proteinase obtained from stem of the pineapple plant (Ananas comosus) and is unique in containing a single oligosaccharide chain attached to the polypeptide. This property allowed its affinity binding and favorable orientation on a Sepharose support pre-coupled with the lectin, concanavalin A (Con A). For comparison, bromelain was also immobilized by covalently coupling to the CNBr-activated Sepharose. The preparation obtained was more resistant to thermal inactivation as evident from the retention of over 50% activity after incubation at 60 for 100 min (as compared to 20% retained by the native enzyme and 30% retained by the covalently immobilized enzyme), exhibited a broader pH-activity profile with the enzyme retaining over 60% activity at pH 11 (as compared to over 25% retained by native and the enzyme immobilized covalently). The native, covalently-coupled and affinity-bound bromelains had apparent K _m values of 1.1, 2 and 0.54 mg/ml, respectively using casein as the substrate. The V _max values remained unaffected on immobilization.     

Some properties of cathepsins chemically fixed to carriers.

An insoluble preparation of rat liver cathepsin D was obtained by coupling the enzyme to Enzacryl Polyacetal (EPA-cathepsin) and to CNBr-activated Sepharose 4B. EPA-cathepsin was active toward the synthetic hexapeptides (Gly-Phe-Leu)2 and did not split hemoglobin. The optimum pH of splitting was displaced upward by 1.5 units to pH 5.0. The enzyme exhibited maximum activity at 60 degrees C. No appreciable loss of activity was seen on storage of the enzyme for 4 months or after repeated use of the preparations. Coupling of rat liver cathepsin D to activated Sepharose gave preparations active towards both protein and synthetic substrates. The preparations were totally inactive in acid media and exhibited maximum activity at pH 7.0, that is, under physiological conditions. Optimum temperature was 65 degrees. The specific activity of the preparations (pH 7.0, 65 degrees) was 60-110 percent that of the free enzyme in acid media. Proteolytic activity of the Sepharose-coupled cathepsin D was not inhibited by pepstatin, whereas that of the free enzyme was fully inhibited by this reagent. A sarcoma cathepsin, similar in some of its properties to the rat liver enzyme, was also coupled to CNBr-activated Sepharose 4B. The preparation split protein substrates at pH 7.0 and possessed enhanced thermostability. The enzymes fixed on Sepharose showed increased stability.     

Purification of IMP dehydrogenase from rat hepatoma 3924A

IMP dehydrogenase (EC 1.1.1.205), the rate-limiting enzyme of de novo GTP biosynthesis and a promising target for cancer chemotherapy, was purified 4860-fold to homogeneity from rat hepatoma 3924A by a method including affinity chromatography in which IMP is bound to epoxy-activated Sepharose 6B. This affinity gel provided a specific elution of the enzyme with 0.5 mM IMP. The final enzyme preparation gave a single band with a molecular weight of 60,000 +/- 1000 on sodium dodecyl sulfate polyacrylamide gel electrophoresis.     

Solid-phase modification with succinic polyethyleneglycol of aminated lipase B from Candida antarctica: Effect of the immobilization protocol on enzyme catalytic properties

Lipase B from Candida antarctica (CALB) has been modified using succinic polyethyleneglycol via the carbodiimide route. Immobilized enzyme (on octyl Sepharose or Eupergit C) has been used, to take advantage of the solid phase. Modification of immobilized CALB's native amino groups did not produce a significant alteration of CALB. However, if the enzyme was previously aminated, around 14–15 PEG molecules could be introduced per enzyme molecule. Also, it has been found that succinic groups are far more reactive than acetic acid following this strategy.Even after this drastic double modification, the functional properties of the enzyme have not been impoverished to a large extent: stability decreased only to some extent (by a 5–6 fold factor), activity versus some substrates even increased (e.g., around 60% using p-nitrophenyl butyrate). It has been found that both modifications (amination and pegylation) have very different effects on enzyme properties when performed on CALB immobilized on Eupergit C or octyl Sepharose. For example, activity versus pNPP increased using CALB-octyl Sepharose while it decreased when using Eupergit C following amination and PEGylation. The effects also depend on the reaction and substrate, for example in hydrolysis of methyl mandelate, the activity decreased by 50% using CALB-octyl Sepharose after PEGylation of the aminated enzyme, while using CALB-Eupergit C had no effect. In this last case, enantioselecitvity in this hydrolysis significantly improved after both chemical modifications (from 7.5 to 20), while using CALB-octyl Sepharose almost had no effect.     

Simultaneous isolation of protein C activator,fibrin clot promoting enzyme (fiprozyme) and phospholipase A2 from the venom of the southern copperhead snake

The simultaneous isolation of three enzymes from the southern copperhead snake venom (Agkistrodon contortrix contortrix; ACC) is described. The first step is a chromatography of crude venom on a Mono S cation-exchange column at pH 6.5. A fibrin clot promoting enzyme (fiprozyme) that preferentially releases fibrinopeptide B from fibrinogen is isolated from the fraction not binding to the Mono S by a further three-step process. The procedure involves affinity chromatography on Blue Sepharose, gel chromatography on Sephacryl S-200 and metal–chelate chromatography on Chelating Sepharose. Protein C activator and phospholipase coelute from the Mono S column. They are separated by a gel chromatography on Sephacryl S-200. After this step two enzymes are obtained: a highly purified protein C activator applicable in methods for determination of functional level of protein C (a plasma regulator of hemostasis) and an electrophoretically pure enzyme with the activity of phospholipase A₂.     

Solubilization and partial purification of squalene synthase from daffodil microsomal membranes

A squalene synthase was solubilized from daffodil (Narcissus pseudonarcissus L.) microsomes with CHAPS, a zwitterionic non-denaturating detergent. By successive chromatography on DEAE Sephacel and APP Sepharose a fraction enriched in this enzyme (21-fold) was prepared.     

A rapid method for the purification of β-lactamase from Bacillus cereus by affinity chromatography

A procedure for the purification of β-lactamase from Bacillus cereus in a single chromatographic step is described. The enzyme is isolated from the crude culture supernatant by affinity chromatography. An inhibitor, methicillin, was immobilised by covalent attachment to the insoluble column gel, Sepharose. The enzyme was adsorbed to the column ligand from the crude supernatant and was subsequently released by increasing the ionic strength of the eluting buffer. In this way the enzyme was selectively isolated from other proteins in the crude supernatant. About 98% of the original β-lactamase activity was recovered in the purified enzyme fraction.     

A simple procedure for isolating adenosine triphosphatase from mitochondria

A simple method for isolation of adenosine triphosphatase (EC 3.6.1.3) from mitochondria is described. The enzyme is released from mitochondrial Lubrol particles by drastic sonication and purified by gel filtration on Sepharose 6-B. The described procedure is effective in isolating adenosine triphosphatase from rat liver as it is from beef heart mitochondria. The enzyme isolated from beef heart has a specific activity of 120 μmol P/min per mg protein and enzyme isolated from rat liver has a specific activity of 70 μmol P/min per mg protein when measured as a release of inorganic phosphate.     

Preparation of Amine Oxidase from Bovine Serum by Affinity Chromatography on Aminohexyl-Sepharose

Amine oxidase was purified from bovine serum by affinity chromatography on aminohexyl substituted Sepharose. The enzyme was adsorbed on the chromatographic support in a suspension of aminohexyl Sepharose in diluted serum. After thorough washing with buffer, the gel was packed in a column and the enzyme eluted with 10 mM octylamine. Using this procedure it was possible to obtain apparently homogenous amine oxidase in a single-step procedure. The specific enzyme activity was 0.14 μoles benzaldehyde formed per minute at 25°C per mg enzyme protein. Based on the activity of amine oxidase in serum, the yield of enzyme was 64 %.     

Rapid purification of adenylate kinase from human erythrocytes and skeletal muscle

Adenylate kinase from human erythrocytes and skeletal muscle can be purified to homogeneity by a new procedure based on DEAE-Sepharose and Blue Sepharose affinity chromatography and Sephadex G-75 fractionation. For the enzyme purified from erythrocytes the specific activity is 3000 U/mg of protein, and the overall yield is 70%. For the enzyme purified from skeletal muscle the specific activity is 2075 U/mg of protein, and the overall yield is 44%. The sequence of steps takes advantage of the high isoelectric point, the high affinity for Blue Sepharose, and the low molecular weight of the isoenzyme from these two human tissues.