Preparation of affinity carriers for the study of binding properties of aspartic proteinases

We have chosen aromatic amino acids and their derivatives as ligands for the affinity chromatography of aspartic proteinases (pepsins and pepsinogens). The following ligands were used: L‐tyrosine, L‐phenylalanine, tyramine, and N‐acetyl‐L‐phenylalanine, and their iodinated derivatives (mono‐ and di‐substituted) with free or blocked amino group. Two types of reactions were used for coupling ligands to Sepharose activated with divinyl sulfone (DVS): via amino group or via carboxyl group. Ligands with free amino group were directly coupled to the activated matrix (L‐tyrosine, 3‐iodo‐L‐tyrosine, 3,5‐diiodo‐L‐tyrosine, L‐phenylalanine, 4‐iodo‐L‐phenylalanine, tyramine); ligands with blocked amino group (N‐acetyl‐L‐phenylalanine, BOC‐L‐tyrosine, BOC‐3,5‐diiodo‐L‐tyrosine; BOC: tert‐butoxy carbonyl) were coupled to Sepharose containing linked ethylenediamine using the carbodiimide reaction. Alternatively, ethylenediamine was bound to free carboxyl croup using the same reaction and these ligand derivatives reacted with divinyl sulfone activated Sepharose. The prepared affinity carriers were used to study the binding properties of porcine pepsin and pepsinogen.     

Application of heptapeptides containing D-amino acid residues immobilized to magnetic particles and Sepharose for the study of binding properties of gastric aspartic proteases

Synthetic heptapeptides containing D-amino acid residues and differing in the content of L-phenylalanine and L-tyrosine residues and their position (Val-D-Leu-Pro-Tyr-Phe-Val-D-Leu, Val-D-Leu-Pro-Tyr-Tyr-Val-D-Leu, Val-D-Leu-Pro-Phe-Tyr-Val-D-Leu) were immobilized to two types of carriers: glyoxal-activated magnetic agarose particles and CNBr-activated Sepharose. In both cases, peptides were immobilized via their terminal amino group. Immobilized peptides were used for the study of binding properties of two gastric aspartic proteases (porcine pepsin A and rat pepsin C). Porcine pepsin A was adsorbed to all studied peptide-modified magnetic carriers, while rat pepsin C interacted with immobilized ligands only slightly. Similar results were obtained in affinity chromatographic experiments using heptapeptides immobilized to Sepharose.     

Optimization of Affinity Chromatography of Pepsins on Iodinated <Emphasis Type="SmallCaps">l</Emphasis>-Tyrosine–Sepharose

Iodinated l-tyrosine–Sepharose was used as an affinity sorbent for the separation of porcine and human pepsins. The optimized conditions for pepsin adsorption as well as enzyme desorption were investigated. The following basic characteristics of the prepared affinity sorbent were determined: the recovery of the loaded enzyme, capacity of the affinity sorbent, concentration range of the linear dependence of bound pepsin on the applied enzyme, and the reproducibility of the experiments. The method described here cannot only be used for pepsin separation but also for analytical purposes.     

Optimization of Affinity Chromatography of Pepsins on Iodinated l-Tyrosine–Sepharose

Iodinated l-tyrosine–Sepharose was used as an affinity sorbent for the separation of porcine and human pepsins. The optimized conditions for pepsin adsorption as well as enzyme desorption were investigated. The following basic characteristics of the prepared affinity sorbent were determined: the recovery of the loaded enzyme, capacity of the affinity sorbent, concentration range of the linear dependence of bound pepsin on the applied enzyme, and the reproducibility of the experiments. The method described here cannot only be used for pepsin separation but also for analytical purposes.

     

Affinity chromatography of porcine pepsin A using quinolin-8-ol as ligand

Stationary phase containing quinolin-8-ol immobilized on macroporous methacrylate support for the affinity chromatography of porcine pepsin A is described. Optimized chromatographic conditions for separation of porcine pepsin A on this stationary phase were found investigating the influence of pH, concentration, ionic strength and chemical composition of the used mobile phases. The stationary phase shows a good reproducibility of chromatographic analyses (relative standard deviation, +/-2%), a high recovery (ca. 93%) and a satisfactory capacity (13 mg pepsin A/1 mL stationary phase) for porcine pepsin A. The obtained findings confirm the applicability of affinity chromatography on the stationary phase with immobilized quinolin-8-ol to the isolation and determination of porcine pepsin A.     

Preparation and testing of stationary phases and modified capillaries for affinity chromatography and affinity capillary electrophoresis of pepsin

Three stationary phases have been prepared for affinity liquid chromatography isolation and separation of porcine and human pepsin. The phases contain 3,5-diiodo-L-tyrosine (DIT) bound to the supports HEMA BIO VS, HEMA BIO E and EPOXY TOYOPEARL. These phases have been tested on a model sample of porcine pepsin A and applied to human pepsin. Fractions have been collected and the chymase activity determined in selected analyses. For affinity CE, capillaries have been prepared by modifying the wall with 3-aminopropyltriethoxysilane, followed either by direct binding of DIT, or by binding L-tyrosine that was subsequently iodated. The dissociation constant K(d) has been determined for the pepsin-DIT complex from the changes in the electrophoretic mobilities.     

Purification and characterization of carbohydrate-binding peptides from Lotus tetragonolobus and Ulex europeus seed lectins using affinity chromatography.

Carbohydrate-binding peptides of several anti-H(O) leguminous lectins were obtained from endoproteinase Asp-N or Lys-C digests of L-fucose-binding Lotus tetragonolobus lectin (LTA) and Ulex europeus lectin I (UEA-I) and from that of a di-N-acetylchitobiose-binding Ulex europeus lectin II (UEA-II) by affinity chromatography on columns of Fuc-Gel (for LTA and UEA-I) and on a column of a mixture of several oligomers of N-acetyl-D-glucosamine (GlcNAc) coupled to Sepharose 4B (GlcNAc oligomer-Sepharose 4B) (for UEA-II). These peptides were retained on the Fuc-Gel or GlcNAc oligomer-Sepharose 4B column and were presumed to have an affinity for the columns. The amino acid sequences of the retarded peptides were determined using a protein sequencer.     

Purification of the pregnancy zone protein by affinity chromatography.

A method for purification of the pregnancy zone protein (PZP) by affinity chromatography was developed. A monospecific immunoglobulin fraction, covalently coupled to Sepharose 4B, was used as binding agent and the elution conditions for PZP are described. The purified protein was shown to have identical properties compared to native PZP with regard to molecular weight, immunodiffusion precipitation and immunosuppressive activity.     

A simplified method for the preparation of WGA-Sepharose 4B and its use in the purification of MN blood group antigens

A simplified method for the preparation of wheat germ agglutinin(WGA)-Sepharose 4B by coupling highly purified WGA, prepared by improved affinity chromatography, with BrCN activated Sepharose 4B in a solution of high carbonate buffer is described. The amount of WGA linked to Sepharose 4B was 82.40% (3.07 mg WGA per ml Sepharose 4B). MN blood group antigens of human erythrocyte membranes purified with WGA-Sepharose 4B affinity chromatography showed a single band on sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS/PAGE). The yield of the antigens from 400 mL fresh blood was 32-40 mg. The WGA-Sepharose 4B column could be used several times without loss of activity.     

Immobilized metal ion affinity electrophoresis. A study with several model proteins containing histidine.

Immobilized metal ion affinity electrophoresis (IMA-Elec) is one among the many methods derived from the immobilized metal ion affinity chromatography. Two approaches for incorporating the metal ligand, were studied. One was in the form of insoluble particulate material based on Sepharose 6B and the other in the form of soluble polymer based on polyethylene glycol (PEG) 5000. Both the polymers coupled with iminodiacetate and metallized with copper or zinc were used as ligands, incorporated into soluble agarose as the electrophoretic gel. Several histidine-containing model proteins were studied with both the systems and their metal binding strengths were determined as the dissociation constants, Kd. The results clearly demonstrated that the mechanism of protein recognition by immobilized copper or zinc via the accessible histidyl residues was maintained in the IMA-Elec system. Proteins with increasing numbers of histidine residues showed increasing binding strength (lower Kd values). While this basic mechanism was conserved, the supporting polymers (Sepharose 6B and the PEG 5000) showed significant differences in the metal binding to the protein. The polysaccharide Sepharose 6B enhanced the binding strength compared with PEG 5000. The optimum electrophoretic parameters were determined to be current intensities up to 20 mA and pH ca. 7.0. At pH greater than 8.0, a significant decrease in the affinity was observed, this decrease being greater with PEG 5000 than Sepharose 6B as supporting material.     

Affinity chromatography of B6-vitamin-dependent enzymes: purification of pig-heart branched-chain amino acid transaminase

Pig-heart branched-chain amino acid transaminase (EC 2.6.1.42) was purified to near homogeneity with a yield of 27%. A prepurification was performed by heat treatment, gel chromatography and DEAE-Sepharose methods. For the final step, several affinity gels were tested and the one containing cycloserine coupled to CNBr-activated Sepharose 4B was selected. This effected an additional five-fold purification with a yield of 60%. The present affinity results are compared with corresponding studies with other aminotransferases in an attempt to find possible universal techniques for their purification.     

Sepharose 4B-DNP-hexamethylenediamine as a sorbent for the chromatography of carboxylic proteinases

Summary 1. The chromatography of the carboxylic proteinases porcine pepsin, aspergillopepsin A, and chymosin on the hydrophobic sorbent Sepharose 4B-DNP-hexamethylenediamine has been studied. It has been shown that the nature of the binding of the proteinases with the sorbent depends on the pH.2. A shortening of the length of the carbohydrate chain of the ligand by four methylene units substantially weakens the interaction of pepsin with the sorbent.3. With chymotrypsin and pepsin as examples, the possibility has been shown of using ionic effects for separating these enzymes.M. V. Lomonosov Moscow State University. Translated from Khimiya Prirodnykh Soedinenii, No. 2, pp. 191–199, March–April, 1979.     

Activation of Supports Containing Hydroxyl Groups Using bis(4-Nitrophenyl) carbonate

Abstract

The hydroxyl groups carrying supports, Sepharose Cl-4B and Fractogel HW75F, were facilely activated under anhydrous conditions using bis(4-nitrophenyl) carbonate (BPNPC), a stable and readily available compound. The activation time at room temperature can be as short as 0.5 hour. The activated supports can be stored at 4°C for at least one month without any loss of coupling capacity. Amines can be coupled to the activated support at pH 7 to 10. Coupling of Protein A to BPNPC activated supports yielded immobilized Protein A affinity supports having a high IgG binding capacity. For example, the Protein A-Sepharose and Protein A-Fractogel prepared by using the BPNPC activation method were able to bind, respectively 13 - 14 mg and 8 - 9 mg human IgG with phosphate buffer saline as the loading buffer. In contrast the use of a high yield binding buffer increases the binding capacity of the two affinity supports to 25 - 28 mg and 22 mg respectively.     

Chromatography of beta-glucuronidase from bovine liver. A study of the enzyme binding sites of prepared adsorbents.

beta-Glucuronidase from bovine liver was adsorbed to the adsorbents prepared with CH-Sepharose 4B and either the competitive inhibitor or its analogs such as p-aminophenyl 1-thio-beta-D-glucuronic acid, -glucoside, -galactoside, and N-acetyl glucosaminide. The adsorbed enzyme was eluted at 0.1 or 0.5 M NaCl by a stepwise gradient. Chromatography of the enzyme was also performed by using the adsorbents prepared with Epoxy-activated Sepharose 6B and amine compounds or other compounds. In order to see whether the hydroxyl groups of the sugar parts in the ligand are necessary for the adsorption of the enzyme, chromatography was performed by using the adsorbents prepared with sugar derivatives as the ligand. As a result, it was found that beta-glucuronidase had an affinity for adsorbents prepared with either acetyl derivatives or methoxy derivatives of glycosides and CH-Sepharose 4B. From the results of elution of the enzyme with NaCl from adsorbents having amide bonding, it was clarified that the affinity of the enzyme for adsorbents without glycosides in the ligands correlated with acidity of the amide in the adsorbents. Hydrogen bond chromatography was performed with the prepared adsorbents. The enzyme was adsorbed under a high concentration of ammonium sulfate, and the elution of the adsorbed enzyme from adsorbents was examined by the degradation of salt. The enzyme was most easily eluted from aminoethyl 1-thio-beta-D-glucuronic acid-CH Sepharose 4B at 0.9 M ammonium sulfate and at 0.5 M concentration of the salt with p-aminophenyl 1-thio-beta-D-glucuronic acid-CH Sepharose 4B.(ABSTRACT TRUNCATED AT 250 WORDS)     

High-performance size-exclusion chromatography of porcine colonic mucins. Comparison of Bio-Gel TSK 40XL and Sepharose 4B columns

A high-performance size-exclusion chromatography (HPSEC) method was developed for the separation of porcine colonic mucins using a Bio-Gel TSK 40XL HPSEC column (300 mm x 75 mm). In addition, porcine gastric and bovine submaxillary mucin preparations were used to describe more fully the separation characteristics of the HPSEC column. For comparison, the same preparations were also separated using a Sepharose 4B column (100 cm x 2.6 cm). The colonic and gastric mucins eluted in the void volume (V0) of both columns. Bovine submaxillary mucin was in the elution volume (Ve) of both columns. Analytical HPSEC of fractions (V0 and Ve) of the various preparations obtained by Sepharose 4B chromatography exhibited retention times identical to those for fractions obtained by HPSEC. After separation by both methods, purified mucins were obtained by CsCl2 density gradient ultracentrifugation; analytical HPSEC profiles, protein contents, and monosaccharide compositions of both gastric and colonic mucins from either column were similar. The HPSEC method, however, is ideally suited to separate microgram to milligram quantities of colonic mucin preparations quickly: 2 to 4 h, compared with 24 to 30 h for the Sepharose 4B method.     

Separation of aspartate aminotransferase from albumin on substituted agaroses

The affinity of aspartate aminotransferase to its inhibitors coupled to Sepharose 4 B was tested. The affinity was measured as retardation of the enzyme compared to “inert” bovine serum albumin. Carboxylic ligands of citrate and 2-oxoglutarate bound to aminoethyl-Sepharose were the best of those tested in separation of the proteins. Because the ligands were not essentially hydrophobic and because it was shown that ion-exchange is not significant in the elution conditions used, it was suggested that the separation is based on the recognition of substrate or effector by the enzyme.     

Modified general affinity adsorbent for large-scale purification of penicillinases

N-Acetyl-D-(-)-penicillamine as a stable second-generation biospecific affinity ligand has previously been suggested for purification of Bacillus cereus 569/H beta-lactamase I. A complex spacer arm is coupled with the matrix by using epichlorohydrin and phloroglucinol doubly activated with divinyl sulphone in the meta position. Coupling of D-(-)-penicillamine ligand resulted in an active affigel. However, we found that two affinity ligands in close proximity prevents simultaneous binding of two penicillinase molecules, therefore one ligand is superfluous. Our results show that: (1) shortening the spacer arm by direct activation of the matrix with divinyl sulphone is satisfactory to produce the affinity material with N-acetyl-D-(-)-penicillamine; (2) incorporation of 15 mumol of N-acetyl-D-(-)-penicillamine per ml of wet Sepharose 4B satisfies the maximum binding capacity requirements of the affigel (about half of the originally incorporated amount of ligand); (3) our simplified affinity adsorbent is generally applicable for large-scale purification of penicillinases to homogeneity from various bacterial sources by the convenient batch method without prior concentration of these enzymes; (4) reacetylation for four/five times can regenerate the original binding capacity of the affigel.     

重组蛋白A亲和填料的制备及其性能评价

为了获得一种优良的抗体纯化介质,制备了重组金黄色葡萄球菌蛋白A(rProtein A)亲和填料,并考察了所制备的亲和填料的纯化性能。利用自行构建的rProtein A工程菌,经诱导表达、纯化获得rProtein A纯品,将其偶联到经环氧氯丙烷活化的Sepharose 4 Fast Flow凝胶上,得到rProtein A亲和填料,并使用兔抗尿酸氧化酶抗体对该填料的性能进行验证。结果显示,在自制的rProtein A亲和填料上rProtein A浓度为1.5×10~4 mol/L。采用Scatchard模型分析,得到其解离常数和最大表观吸附量分别为2.28×10~7 mol/L和20.697 g/L,说明制得的rProtein A亲和填料对抗体有很好的结合能力。将该填料于0.1 mol/L NaOH溶液中浸泡1 h,其色谱性能未见变化。将该填料用于纯化兔抗体,湿胶结合抗体量可达19 mg/mL;一步柱色谱即可得到电泳纯度的抗体样品,回收率高于96%。本研究为rProtein A亲和填料的国产化奠定了基础。     

Favourable biospecific reactivity of blood group B antigenic trisaccharide chemically attached to poly-N-(2-hydroxyethyl)acrylamide-coated porous glass.

Blood group B antigenic trisaccharide-beta-aminopropyl glycoside (B-TSAP) covalently attached to poly-N-(2-hydroxyethyl)acrylamide-coated porous glass interacts with anti-B monoclonal antibodies faster than the ligand coupled to CNBr-activated Sepharose 4B and Affi-Gel 10. Rates of hydrophobic adsorption of antibodies on the butyl derivatives of the same supports were measured to evaluate the diffusion input to overall kinetics. The lowest average affinity adsorption time [t1(aff) = 250 s] observed for polymer-coated glass probably arises because of the flexibility of the extended segments of chemisorbed N-substituted polyacrylamide acting as effective spacer arms.     

Affinity Chromatography of Insulin with a Heptapeptide Ligand Selected from Phage Display Library

A heptapeptide phage display library was screened with insulin to find its ligands for affinity chromatography. The peptide was synthesized and coupled to EAH Sepharose 4B (5.4 mol mL^–1 bed). Then, insulin chromatography was carried out with mobile phases of different pH values and by the addition of urea and ethylene glycol. It was found that electrostatic interactions were predominant for the affinity binding, and hydrogen bonding might also contribute somewhat to the affinity. Finally, frontal analysis was performed and the dynamic binding capacity of the affinity column for insulin at 50% breakthrough was estimated at 60.6 mg mL^–1 bed, which was about two times higher than the theoretical binding capacity of the monomeric insulin. The result suggests that insulin was bound in dimer state in a stoichiometric relationship with the coupled peptide, indicating the high binding efficiency of the peptide ligand for insulin.