Kinetics of immobilized pig heart fumarase

The kinetics of immobilized pig heart fumarase are described and compared with the properties of the enzyme free in solution.

1. An analogous pH dependence of initial activity is found for free and immobilized enzyme.
2. Immobilized and free fumarase deviate from classical Michaelis-Menten kinetics in the same way. The apparent K_m values are three to eight times higher for the immobilized (2 mg/g gel) enzyme.
3. The specific activity of immobilized fumarase is dependent on the final enzyme concentration on the gel; normal specific activities are observed when 50 ?g fumarase is immobilized per gram of gel, whereas the specific activity decreases with increasing enzyme concentration.
4. The activation energies for free and immobilized fumarase (50 ?g/g gel) were found to be identical between 22 and 32?C and with L-malate as substrate (E_a = 12,290 cal/mol at pH 7.9). Upon increasing the concentration of fumarase on the gel, the activation energy decreases.

Our results indicate that the true catalytic properties of fumarase are not affected by immobilization of this enzyme. The slight differences observed when fumarase is immobilized at concentrations higher than 50 ?g/g gel must be attributed to diffusional limitation at the surface of the Sepharose matrix.     

Rapid purification of antisteroid antibodies by high-performance liquid affinity chromatography

An adsorbent for the high-performance affinity chromatography of antisteroid antibodies was prepared, based on a commercial pre-packed column. The column contained activated microparticulate silica beads bearing epoxide functions, on which the steroid dexamethasone was covalently linked. The column was used successfully for the rapid and complete isolation of several hundred microgram amounts of specific antidexamethasone antibodies from rabbit antisera. The practical aspects of the purification procedure, especially the optimization of the washing and of the elution steps, are detailed. Despite non-biospecific elution with 20% acetonitrile in an acidic buffer, the purification yield was very satisfactory and the biological activity of the purified immunoglobulins appeared excellent.     

Thiacarbocyanine as ligand in dye-affinity chromatography for protein purification

A thiacarbocyanine has been post-grafted onto beaded cellulose by a curing method envisioning their use as a biomimetic ligand in dye-affinity chromatography. The immobilization of this dye was performed based on a previously brief derivatization study where the analogous microcrystalline cellulose was alternatively used and some of the curing reaction conditions were varied. The grafted beaded cellulose so obtained was qualitatively and quantitatively characterized by SEM, SEM-EDS and EA. The immobilized dye-affinity interaction with the standard proteins bovine serum albumin, alpha-chymotrypsin and lysosyme was analysed. The influence of the mobile phase composition on the chromatographic behaviour of these standard proteins was also studied. A selective interaction was observed allowing the separation of all the three proteins from an artificial mixture.     

Isolation and purification of monosialotetrahexosylgangliosides from pig brain by extraction and liquid chromatography

Monosialotetrahexosylganglioside (GM1), one of glycosphingolipids containing sialic acid, plays particularly important role in fighting against paralysis, dementia and other diseases caused by brain and nerve damage. In this work, a simple and highly efficient method with high yield was developed for isolation and purification of GM1 from pig brain. The method consisted of an extraction by chloroform–methanol–water and a two‐step chromatographic separation by DEAE–Sepharose Fast Flow anion‐exchange medium and Sephacryl S‐100 HR size‐exclusion medium. The purified GM1 was proved to be homogeneous and had a purity of >98.0% by high‐performance anion‐exchange and size‐exclusion chromatography. The molecular weight was 30.0 kDa by high‐performance size‐exclusion chromatography and 1546.9 Da by electrospray ionization mass spectrometry. The chromogenic reaction by resorcinol–hydrochloric acid solution indicated that the purified GM1 showed a specific chromogenic reaction of sialic acid. Through this isolation and purification program, ~1.0 mg of pure GM1 could be captured from 500 g wet pig brain tissue and the yield of GM1 was around 0.022%, which was higher than the yields by other methods. The method may provide an alternative for isolation and purification of GM₁ in other biological tissues. Copyright © 2015 John Wiley & Sons, Ltd.     

Rapid purification of the main allergen of Lolium perenne by high-performance liquid chromatography

The main allergen from rye grass (Lolium perenne) pollen was purified by size-exclusion high-performance liquid chromatography. The purified allergen had a molecular weight of 32,000 daltons and was significantly more active in solid phase radioimmunoassay than the whole extract. The highly purified antigen can be obtained very rapidly and with a recovery of 30%.     

Rapid and efficient purification of positron emission tomography probes by hydrophilic interaction chromatography

A rapid and efficient preparative high-performance liquid chromatographic procedure was established to purify short-lived positron emission tomography radio-probes. This method is based on hydrophilic interaction chromatography utilizing a semi-preparative silica column (10 mm I.D.) and a high volatile organic mobile phase (>90% acetonitrile). In nine different radiopharmaceuticals studied, six compounds could be separated from the unlabeled precursor with good resolution and faster elution than its precursor. These characteristics enabled significant shortening of the separation and evaporation processes in the manufacture of short-lived radiopharmaceuticals. Several ¹¹C-radiopharmaceuticals could be prepared within one half-life of carbon-11 (20.4 min), including radiosynthesis, purification and formulation steps with sufficient radiochemical/chemical purity and high levels of radioactivity/specific radioactivity.     

Rapid purification of detergent-solubilized bovine hormone-sensitive lipase by high performance hydrophobic interaction chromatography

Hormone-sensitive lipase (HSL), the enzyme controlling the rate of adipose tissue lipolysis and also possibly involved in the regulation of steroidogenesis, has been purified from bovine omental adipose tissue. Partially detergent-solubilized, delipidated and purified HSL was obtained through step-elution at conventional DEAE ion-exchange chromatography, followed by concentration on hydroxylapatite. High performance hydrophobic interaction chromatography (HPHIC) on phenylsilica then resulted in an increase of HSL protein purity from 2% to more than 70%. Final purification of the enzyme to apparent homogeneity (greater than 95% protein purity), concentration and removal of most of the detergent was obtained by high performance cation exchange chromatography on Mono S. At least 0.5 mg of highly stable HSL was obtained from 5 kg of bovine omental fat within four working days. The purified lipase had a lower specific activity than previously reported for the corresponding rat enzyme but the preparations have proved very useful for enzyme structure studies and as an antigen.     

Understanding ligand–protein interactions in affinity membrane chromatography for antibody purification

Affinity chromatography with Protein A beads has become the conventional unit operation for the primary capture of monoclonal antibodies. However, Protein A activated supports are expensive and ligand leakage is an issue to be considered. In addition, the limited production capabilities of the chromatographic process drive the research towards feasible alternatives. The use of synthetic ligands as Protein A substitutes has been considered in this work. Synthetic ligands, that mimic the interaction between Protein A and the constant fragment (Fc) of immunoglobulins, have been immobilized on cellulosic membrane supports. The resulting affinity membranes have been experimentally characterized with pure immunoglobulin G (IgG). The effects of the membrane support and of the spacer arm on the ligand–ligate interaction have been studied in detail. Experimental data have been compared with molecular dynamic simulations with the aim of better understanding the interaction mechanisms. Molecular dynamic simulations were performed in explicit water, modelling the membrane as a matrix of overlapped glucopyranose units. Electrostatic charges of the ligand and spacer were calculated through ab initio methods to complete the force field used to model the membrane. The simulations enabled to elucidate how the interactions of surface, spacer and ligand with IgG, contribute to the formation of the bond between protein and affinity membrane.     

Investigation of enzyme-substrate complexes by affinity chromatography. Application to pig heart citrate synthase

The relative affinities of Sepharose gels, to which coenzyme A (CoA-SH) and CoA-SH analogues were bound through a well defined site, for citrate synthase were determined. The relative eluting power of coenzyme derivatives for the CoA-SH-gel and the Matrex Gel Blue-bound enzyme was measured, and the influence of oxaloacetate on the binding of the enzyme investigated. From the results, the contributions of different parts of the coenzyme to its binding in the active site and kinetic concepts are derived and found to be in complete agreement with corresponding data for citrate synthase obtained from kinetic measurements reported in the literature. It is demonstrated for some other CoA-SH-specific enzymes that affinity chromatography is of value as an additional tool for the comparative investigation of binding sites of enzymes which depend on the same coenzyme.     

Sepharose derivatives containing citric acid as affinity ligand. Purification of fumarase

Six Sepharose derivatives, in which citrate was immobilized via methylene carbons, were prepared by coupling of the alpha- and beta-isomers of citrylpolymethylenediamine to Sepharose. The purification of fumarase from pig heart was dependent on the length of the spacer arm, but not on the isomeric configuration of the immobilized citrate. Gels having six methylene carbons had the largest adsorption capacity for the enzyme and therefore were the most suitable for use in affinity columns for its purification. Affinity chromatography with these gels was followed by hydrophobic interaction chromatography on an octamethylenediamine-Sepharose column.     

Rapid purification and scale-up of honokiol and magnolol using high-capacity high-speed counter-current chromatography

In this paper, a rapid separation approach has been developed using high-capacity high-speed counter-current chromatography (high-capacity HSCCC) to isolate and purify honokiol and magnolol, which are the main bioactive constituents from Houpu. The optimization of the solvent selection process, sample loading volume and flow rate is systematically studied using analytical high-capacity HSCCC. The optimized parameters obtained rapidly at analytical scale were used for a 1000 x scale-up preparative run using pilot scale high-capacity HSCCC in a MAXI-DE centrifuge. A crude sample of 43 g was successfully separated and the fractions were analysed by high-performance liquid chromatography (HPLC). This large scale preparative single step run yielded 16.9 and 19.4 g of honokiol and magnolol with purities of 98.6 and 99.9%, in only 20 min. This is the first time that high-performance counter-current chromatography has been used to purify multiple gram grade bioactive compounds in less than 1h and at such high concentrations of final products (10.8 g/l for magnolol and 7.0 g/l for honokiol).