Trypsin and affinity chromatography.

Affinity adsorbents for trypsin which were prepared by immobilizing product-type ligands, that is, peptides having C-terminal arginine, proved to be effective not only for preparative purposes but also for basic research on molecular recognition. The properties of the binding site of trypsin were revealed by chromatographic experiments. Quantitative analysis based on the theory of frontal affinity chromatography proved to be extremely effective. As an extension of the product-type ligands, peptide argininals were also used and information on the mechanism of action of these inhibitors was obtained. Anhydrotrypsin, which lost the hydroxyl group of Ser183, was found to gain increased binding ability for product-type compounds. This inactivated enzyme was also used as an immobilized ligand and the unique affinity adsorbent thus prepared proved to be extremely effective for the separation of peptides and recombinant proteins based on their C-terminal structures. High-performance affinity chromatography of trypsin and related enzymes using a polymer-based support was also developed.     

Strategy for the isolation of native dehydrogenases with potential for biosensor development from the organism Hyphomicrobium zavarzinii ZV580

Dehydrogenases are interesting candidates for the development of electrochemical biosensors. Most dehydrogenases are characterised by a comparatively broad substrate spectrum, yet highly specific enzymes exist as well. A specific formaldehyde dehydrogenase has, e.g., been described for the organism Hyphomicrobium zavarzinii ZV580. Isolation of enzymes from their natural source instead of a recombinant expression renders the isolation more challenging, as common tools such as affinity tags are no longer available. In this contribution, we develop chromatographic procedures for such isolation tasks. The previously described formaldehyde dehydrogenase was isolated by two procedures, one based on affinity chromatography, the other on hydroxyapatite. Neither procedure yielded an active enzyme. In addition two dehydrogenases, a formaldehyde and a methylamine dehydrogenase, were found in the cell free extract, which had not been described previously. Both enzymes could be isolated to near purity by a sequence of hydroxyapatite and anion exchange chromatography. The new formaldehyde dehydrogenase requires reconstitution with calcium and pyrroloquinoline quinone in order to become active. The enzyme shows no cross-reactivity with methylamine or methanol. The methylamine dehydrogenase catalyses the conversion of methylamine into formaldehyde, hence it could become a technical catalyst for the inverse reaction. This enzyme consists of two types of subunit and may be one of the rare alpha,beta-methylamine dehydrogenases.     

Study of affinity supports based on reactive polymers immobilized on silica: affinity constant determination from isocratic zonal elution.

Polymers bearing benzamidine moieties have been prepared from reactive copolymer containing chloroformate functions and deposited on porous silica matrices. These high-performance affinity chromatography supports were characterized by quantitative methods, which analyse the zonal elution behaviour of trypsin in the presence of a soluble competitor (L-arginine). The column loading capacity for trypsin was measured by the zonal elution method in mass overload conditions. On the basis of a Langmuir isotherm, the influence of the protein capacity and the concentration of the soluble ligand on the elution volume was studied for the determination of the binding constants. The plate heights determined for silica supports of various porosities and particle diameters show that the strong affinity interactions between trypsin and p-aminobenzamidine are mainly responsible for the low efficiencies observed.     

In flow activation of diol-silica with cyanogen bromide and triethylamine for preparing high-performance affinity chromatographic columns

A new coupling strategy using pre-packed diol-silica supports to obtain affinity columns for high-performance affinity chromatography (HPAC) is described. These columns were prepared by "in flow" activation in which solutions containing anhydrous solutions of CNBr and triethylamine are separately pumped to a mixer and then onto a pre-packed diol-silica column. Recycling the amino ligand to be coupled several times over the activated silica diol columns results in ligand immobilization. DNA (the Op 1 lac operator), 6-aminohexyl-Cibacron and a peptide (melittin) were all successfully "in flow" coupled to freshly activated columns. Methods for CNBr activation of pre-packed diol-silica column were developed for one, two or three pump HPLC systems. The supports were successfully used for the HPAC purification of a Lac repressor-beta-galactosidase fusion protein, alcohol dehydrogenase, and calmodulin. Columns prepared by in flow activation/coupling procedures were shown to be stable for at least 14 months. Also, in flow activated silica columns could be stored in anhydrous acetone for at least 3 months prior to coupling. Our experiments with these affinity ligand columns (DNA-silica, aminohexyl-Cibacron F3GA-silica, and melittin-silica), suggests that this is a very successful coupling protocol for producing a variety of HPAC columns.     

Applications of silica supports in affinity chromatography

The combined use of silica-based chromatographic supports with immobilized affinity ligands can be used in many preparative and analytical applications. One example is the use of silica-based affinity columns in HPLC, giving rise to a method known as high-performance affinity chromatography (HPAC). This review discusses the role that silica has played in the development of affinity chromatography and HPAC and the applications of silica in these methods. This includes a discussion of the types of ligands that have been employed with silica and the methods by which these ligands have been immobilized. Various formats have also been presented for the use of silica in affinity chromatographic methods, including assays involving direct or indirect analyte detection, on-line or off-line affinity extraction, and chiral separations. The use of silica-based affinity columns in studies of biological systems based on zonal elution and frontal analysis methods will also be considered.     

Affinity chromatography of fibroblast growth factors on coated silica supports grafted with heparin.

Dextran-coated silica beads are excellent supports for affinity chromatography of proteins. They can be easily grafted using conventional coupling methods with different active ligands, such as heparin. Fibroblast growth factors develop specific interactions with heparin through well-defined amino acids sequences. The heparin-dextran coated silica phases exhibit an affinity for these growth factors. Under our experimental conditions, the basic form can be absorbed on the solid support at a moderate salt concentration (0.5 M sodium chloride) and can be selectively desorbed by increasing the ionic strength of the eluent. The purification performances of such phases are compared to those obtained on the heparin grafted soft gels. Because of their mechanical properties, the dextran-coated silica supports were also used in high-performance affinity chromatography to purify fibroblast growth factors from a bovine brain crude extract.     

Preparation of anhydrotrypsin-immobilized diol silica as a selective adsorbent for high-performance affinity chromatography of peptides containing arginine or lysine at their C-termini

Summary Anhydrotrypsin (AHT), a catalytically inert derivative of trypsin in which the active site serine residue was converted to dehydroalanine residue by chemical modification, was immobilized onto diol silica through the activation with trifluoroethanesulfonyl chloride, and an AHT-diol-silica column was used for high-performance affinity chromatography separation of peptides containing arginine or lysine at their C-termini from the others. Improved separation in terms of speed was accomplished.     

Fast Affinity Chromatography Using Small Particle Silica-Based Packing Materials

Summary

Affinity chromatography is one of the most powerful techniques for the purification of biologically active proteins available (for review see [1]). The ability of this method to purify proteins is based on highly specific, selective or characteristic interactions with immobilized ligands. Several advantages over traditional soft gel affinity supports have been observed with the use of small particle silica based materials for high performance affinity chromatography. These include greatly improved mass transfer properties which allow separations that are not always practical in the low performance mode, greatly reduced equilibration and isolation times, high available ligand densities, small elution volumes, excellent recovery of very small quantities of protein and high dynamic capacities. The criteria for developing a general, derivatizable, high performance support for high performance affinity chromatography are discussed. The step-by-step examination of these criteria and experimental evidence for determining parameters such as ligand density, non-specific adsorption and column life time for such a system are described. Chromatographic results are shown for preparative separations of (i) receptor proteins, (ii) antibodies and (iii) active enzymes.     

A strategy for screening trypsin inhibitors from traditional Chinese medicine based on a monolithic capillary immobilized enzyme reactor coupled with offline liquid chromatography and mass spectrometry

A novel strategy was successfully developed for screening trypsin inhibitors in traditional Chinese medicines based on monolithic capillary immobilized enzyme reactors combined with liquid chromatography‐tandem mass spectrometry. Organic polymer based monolithic enzyme reactors were firstly prepared by covalently bonding trypsin to a poly(glycidyl methacrylate‐co‐poly (ethylene glycol) diacrylate) monolith by the ring‐opening reaction of epoxy groups. The activity and kinetic parameters of the obtained monolithic trypsin reactors were systematically evaluated using micro‐liquid chromatography. Fourier transform infrared spectroscopy and scanning electron microscopy were also used to characterize the monolithic trypsin reactors. The resulting functional and denatured monolithic trypsin reactors were applied as affinity solid‐phase extraction columns, and offline coupled with a liquid chromatography‐tandem mass spectrometry system to construct a binding affinity screening platform. Subsequently, the proposed platform was applied for screening trypsin binders in a Scutellaria baicalensis Georgi extract. Three compounds, namely scutellarin, baicalin, and wogonoside were identified, and their inhibitory activities were further confirmed via an in vitro enzymatic inhibition assay. Additionally, molecular docking was also performed to study the interactions between trypsin and these three compounds.     

Characterization of p-aminobenzamidine-based sorbent and its use for high-performance affinity chromatography of trypsin-like proteases

An affinity sorbent, hydrophilic polymer-based carrier of different pore size (Toyopearl) with immobilized p-aminobenzamidine (ABA), has been prepared. Its basic properties and some applications for protein purification were studied. ABA, which is a synthetic inhibitor for trypsin-like proteases, was covalently immobilized to Toyopearl by reductive amination. The ligand density and binding capacity for porcine trypsin varied depending on the pore size of Toyopearl. The maximum binding capacity of the immobilized p-aminobenzamidine Toyopearl (ABA-Toyopearl) for trypsin was more than 40 mg/ml gel. ABA-Toyopearl thus obtained was very stable below pH 8 and was successfully used for high-performance affinity chromatography of trypsin-like proteases such as trypsin, thrombin, tissue-type plasminogen activator or urokinase in a single step at 25 degrees C.     

Adsorption of trypsin on hydrophilic and hydrophobic surfaces

The adsorption of trypsin onto polystyrene and silica surfaces was investigated by reflectometry, spectroscopic methods, and atomic force microscopy (AFM). The affinity of trypsin for the hydrophobic polystyrene surface was higher than that for the hydrophilic silica surface, but steady-state adsorbed amounts were about the same at both surfaces. The conformational characteristics of trypsin immobilized on silica and polystyrene nanospheres were analyzed in situ by circular dichroism and fluorescence spectroscopy. Upon adsorption the trypsin molecules underwent structural changes at the secondary and tertiary level, although the nature of the structural alterations was different for silica and polystyrene surfaces. AFM imaging of trypsin adsorbed on silica showed clustering of enzyme molecules. Rinsing the silica surface resulted in 20% desorption of the originally adsorbed enzyme molecules. Adsorption of trypsin on the surface of polystyrene was almost irreversible with respect to dilution. After adsorption on silica the enzymatic activity of trypsin was 10 times lower, and adsorbed on polystyrene the activity was completely suppressed. The trypsin molecules that were desorbed from the sorbent surfaces by dilution with buffer regained full enzymatic activity.     

Purification and partial characterization of the pancreatic proteolytic enzymes trypsin, chymotrypsin, and elastase from the chicken.

The purpose of this work was to isolate, purify and partially sequence trypsin, chymotrypsin and elastase from the chicken pancreas. The extraction of the pancreatic zymogens with 0.5 M CaCl2 at pH 7.5 for 9 h appeared to be most effective in obtaining maximum recovery of the three enzymes. The sequential Cucurbita maxima trypsin inhibitor I/bovine pancreas trypsin inhibitor/soybean trypsin inhibitor affinity chromatography gave the best result for the isolation of trypsin, chymotrypsin and elastase, respectively, from the same extract. For each proteinase, multiple form of enzymatic activity could be observed after gel electrophoresis and each form was further purified on an ion-exchange column. The N-terminal amino acid sequence of trypsin and chymotrypsin showed homologies with the bovine enzymes whereas elastase showed homologies with the porcine enzyme. The molecular mass of trypsin, chymotrypsin and elastase were estimated to be 23,500, 25,700 and 25,000, respectively, which are values close to those in mammalian species. Although some kinetic constants (Km and k(cat)/Km) appeared different from those observed in other species, the pH dependent enzymatic activities were similar to those reported in other animal species.     

Studies on Synthesis and Chromatographic Properties of NAD Covalently Bound onto Phospholipid-Coated Aminated Silica

Abstract

Recently affinity chromatography with a biospecific stationary phase was widely used in separation and purification processes of all kinds of enzymes. In this paper, a series of synthetic reactions of solid-liquid phase on a silica surface are described. By using a wide-pore (30μm). microspheric silica (8μm) as the matrix and γ-aminopropyltriethoxy-silane as the activating agent, the nicotinamide adenine dinucleotide (NAD) was bonded through its amino groups with carboxylic groups of linked phospholipid by a covalent bond on the aminated supports. This bonded stationary phase provided high thermal stability which could be used for separating nucleotides with good resolution. At the same time, the effects of pH, organic modifier and ionic strength on the retention properties of nucleotides were also investigated.     

Affinity monolith chromatography: a review of principles and recent analytical applications

Affinity monolith chromatography (AMC) is a type of liquid chromatography that uses a monolithic support and a biologically related binding agent as a stationary phase. AMC is a powerful method for the selective separation, analysis, or study of specific target compounds in a sample. This review discusses the basic principles of AMC and recent developments and applications of this method, with particular emphasis being given to work that has appeared in the last 5 years. Various materials that have been used to prepare columns for AMC are examined, including organic monoliths, silica monoliths, agarose monoliths, and cryogels. These supports have been used in AMC for formats that have ranged from traditional columns to disks, microcolumns, and capillaries. Many binding agents have also been employed in AMC, such as antibodies, enzymes, proteins, lectins, immobilized metal ions, and dyes. Some applications that have been reported with these binding agents in AMC are bioaffinity chromatography, immunoaffinity chromatography or immunoextraction, immobilized-metal-ion affinity chromatography, dye–ligand affinity chromatography, chiral separations, and biointeraction studies. Examples are presented from fields that include analytical chemistry, pharmaceutical analysis, clinical testing, and biotechnology. Current trends and possible directions in AMC are also discussed.     

Protein purification by aminosquarylium cyanine dye‐affinity chromatography

The most selective purification method for proteins and other biomolecules is affinity chromatography. This method is based on the unique biological‐based specificity of the biomolecule–ligand interaction and commonly uses biological ligands. However, these ligands may present some drawbacks, mainly because of their cost and lability. Dye‐affinity chromatography overcomes the limitations of biological ligands and is widely used owing to the low cost of synthetic dyes and to their resistance to biological and chemical degradation. In this work, immobilized aminosquarylium cyanine dyes are used in order to exploit affinity interactions with standard proteins such as lysozyme, α‐chymotrypsin and trypsin. These studies evaluate the affinity interactions occurring between the immobilized ligand and the different proteins, as a reflection of the sum of several molecular interactions, namely ionic, hydrophobic and van der Waals, spread throughout the structure, in a defined spatial manner. The results show the possibility of using an aminosquarylium cyanine dye bearing a N‐hexyl pendant chain, with a ligand density of 1.8 × 10^?2 mmol of dye/g of chromatographic support, to isolate lysozyme, α‐chymotrypsin and trypsin from a mixture. The application of a decreasing ammonium sulfate gradient resulted in the recovery of lysozyme in the flowthrough. On the other hand, α‐chymotrypsin and trypsin were retained, involving different interactions with the ligand. In conclusion, this study demonstrates the potential applicability of ligands such as aminosquarylium cyanine dyes for the separation and purification of proteins by affinity chromatography. Copyright © 2013 John Wiley & Sons, Ltd.     

单分散非多孔交联聚甲基丙烯酸环氧丙酯微球作为亲和色谱载体的研究

单分散、非多孔聚甲基丙烯酸环丙酯生球机械强度高,表面的环基易于修饰与键合,非常适宜于生物大分子的快速分离、纯化。对其表面环氧基浓度、非特异性地性能等进行了表征;并以胰蛋白酶抑制剂为配基,制备了胰蛋白酶抑制剂－交联聚甲基丙烯权环氧丙酯亲和色柱,考察了胰蛋白酶等蛋白南的亲和色谱行为,表明该基质适宜用作亲和色谱载体。提出以添加１％乙腈的方法,可有癣地消除此类载体的弱非特异性吸附,使人血清白蛋白的回收率     

Rigid Support Materials for the Immobilization of Enzymes

The purpose of the work presented here was to prepare a support material for enzymes and “affinity ligands” with the following characteristics: low cost, durability, rigidity, and high capacity. Our study encompassed conjugates of porous and nonporous silicas with organic polymers and macroporous ion-exchange resins. Poly-ethyleneimine (PEI), polyacrylic acid (PAA), poly (methyl vinyl ether/maleic anhydride) were attached to porous glass and silica in various combinations. The composite of silica beads with PEI and PAA is a good support for the enzyme trypsin as judged by the activity against N-α-benzoyl-L-arginine ethyl ester.

Amberlyst (macroporous, sulfonated polystyrene) was activated by treatment with thionyl chloride; the resulting resin was either used directly or reacted with a diamine. The diamine derivative was used for enzyme coupling or transformed further to the succinyl or p-aminobenzoyl derivative. None of these derivatives were particularly good as supports for the enzyme trypsin. Duolite converted to a PAA, succinyl, or succinimide derivative was a good support. The enzyme-resin adduct has good activity and stability.

The resin is quite durable and of low cost. The Duolite-trypsin has good activity against protein. In addition, this derivative was active in 7 M urea. The proteolytic activity was nearly doubled by urea, presumably as a result of substrate (casein) denaturation. The michaelis constants and pH dependences are compared for trypsin conjugates with Duolite A-7, Silica-PEI-PAA, agarose, and porous glass. A cost comparison reveals that the Duolite and silica derivatives are much less expensive than agarose and glass.     

Evaluation and optimization of the metal‐binding properties of a complex ligand for immobilized metal affinity chromatography

The simultaneous determination of two binding parameters for metal ions on an immobilized metal affinity chromatography column was performed by frontal chromatography. In this study, the binding parameters of Cu²⁺ to l ‐glutamic acid were measured, the metal ion‐binding characteristics of the complex ligand were evaluated. The linear correlation coefficients were all greater than 99%, and the relative standard deviations of two binding parameters were 0.58 and 0.059%, respectively. The experiments proved that the frontal chromatography method was accurate, reproducible, and could be used to determine the metal‐binding parameters of the affinity column. The effects of buffer pH, type, and concentration on binding parameters were explored by uniform design experiment. Regression, matching and residual analyses of the models were performed. Meanwhile, the optimum‐binding conditions of Cu²⁺ on the l ‐glutamic acid‐silica column were obtained. Under these binding conditions, observations and regression values of two parameters were similar, and the observation values were the best. The results demonstrated that high intensity metal affinity column could be effectively prepared by measuring and evaluating binding parameters using frontal chromatography combined with a uniform design experiment. The present work provided a new mode for evaluating and preparing immobilized metal affinity column with good metal‐binding behaviors.     

Immobilized trypsin systems coupled on-line to separation methods: recent developments and analytical applications

The ability to rapidly and efficiently digest and identify an unknown protein is of great utility for proteome studies. Identification of proteins via peptide mapping is generally accomplished through proteolytic digestion with enzymes such as trypsin. Limitations of this approach consist in manual sample manipulation steps and extended reaction times for proteolytic digestion. The use of immobilized trypsin for cleavage of proteins is advantageous in comparison with application of its soluble form. Enzymes can be immobilized on different supports and used in flow systems such as immobilized enzyme reactors (IMERs). This review reports applications of immobilized trypsin reactors in which the IMER has been integrated into separation systems such as reversed-phase liquid chromatography or capillary electrophoresis, prior to MS analysis. Immobilization procedures including supports, mode of integration into separation systems, and methods are described.     

Separation of immunoglobulin G by high-performance pseudo-bioaffinity chromatography with immobilized histidine. I. Preliminary report on the influence of the silica support and the coupling mode.

High-performance liquid affinity chromatography with immobilized histidine as a pseudo-biospecific ligand has been used for the fractionation of human immunoglobulin G (IgG). Histidine was immobilized onto silica in two different modes: directly onto silica after epoxy activation or using an intermediate amino derivatization of silica and then coupling histidine using water-soluble carbodiimide. The behaviours and capacities of the obtained affinity supports as well as the influence of pH, silica type, pore diameter and coupling mode have been studied. IgG was effectively separated from human plasma and high maximal binding capacities were obtained.