Affinity chromatography of the muscarinic acetylcholine receptor

A novel compound, 3-(2'-aminobenzhydryloxy)-tropane (ABT), and an ABT-agarose gel were synthesized and used for the purification of solubilized muscarinic receptors. ABT had a high affinity with an apparent dissociation constant (Kd) of 7 nM for the muscarinic receptors solubilized from the porcine brain by digitonin. An ABT-agarose gel was prepared by coupling ABT with epoxy-activated Sepharose 6B, and the degree of substitution to the gel was determined to be 4-5 mumol/ml of the gel by UV absorption spectrum. During affinity chromatography using 10 ml of the ABT-agarose gel and 100 ml of the digitonin-solubilized preparation, 70% of muscarinic receptors were adsorbed to the gel, in marked contrast with the adsorption of only 2% of proteins. Approximately 25% of muscarinic receptors applied to the gel were eluted biospecifically with 1 mM muscarinic ligands. The purified fraction showed a high affinity for [3H]quinuclidinyl benzylate with a Kd of 0.4 nM and similar specificity for muscarinic ligands to that of unpurified soluble receptors. The protein concentration of the purified fraction was too low to be determined accurately, but very approximately a purification of 10(3)-fold was indicated.     

Application of adsorption kinetics for estimation of dissociation constants

Substances such as drugs, as well as special ligands with expressive biospecific properties, all with different affinities, interact with proteins which can be characterized by dissociation constants. The method for estimation of the dissociation constant on the basis of adsorption kinetics was verified for two typical cases: adsorption of lactate dehydrogenase onto bead cellulose derivatized by reactive dyes C.I.2. or C.I.19, and adsorption of different drugs (neuroleptics and local anesthetics) onto calmodulin immobilized on agarose gel. The real equilibrium values obtained by using the complete time-concentration model of adsorption were fitted according to the respective adsorption isotherms by non-linear regression.     

魔竽葡苷聚糖凝胶为亲和层析载体与Sepharose 4B的比较研究——Ⅰ.KGM金属螯和层析分离纯化猪血SOD

将KGM凝胶和Sepharose 4B在同样条件下活化偶联,制成Cu~(2 )金属螫合亲和胶,亲和纯化猪血SOD,并对这两种亲和胶的层析效果和性能进行了比较。KGM金属螫合胶对猪血SOD吸附量、纯化倍数、纯化SOD的比活力和回收率分别为53000U/ml胶、19倍、12000U/mg蛋白和94.6％,而Sepharose 4B亲和胶对SOD 的吸附量、纯化倍数、纯化SOD的比活力和回收率分别为79920U/ml胶、11倍、10125U/mg蛋白和95.4％。两种亲和胶所纯化的SOD经聚丙烯酰胺凝胶电泳(PAGE)、活性染色及SDS聚丙烯酰胺凝胶电泳(SDS-PAGE)证明其均为电泳纯。KGM金属螯合胶使用六次后,其对SOD吸附量、去Cu量及SOD的回收率均无明显影响。     

Multiple-point adsorption of terbium ions by lead ion templated thermosensitive gel: elucidating recognition of conformation in gel by terbium probe

Lead ion templated thermosensitive heteropolymer gel which has recognition ability of methacrylate pairs has been synthesized and characterized. The gel consists of a main monomer component, N-isopropylacrylamide (NIPA), responsible for volume phase transition, methacrylic acid (MAA) moieties imprinted as pairs to adsorb terbium ions and cross-links. An imprinting technique was applied using lead ion complex with methacrylate ligands in dioxane media. After gel was obtained, lead ions were removed by washing and the imprinted gel showed strong binding ability to terbium ions, comparable with that of the non-imprinted gel prepared without lead ions. It was found that the Tb(3+) fluorescence intensity was considerably increased upon binding this ion to both imprinted and non-imprinted gels, but the largest enhancement of fluorescence intensity was observed when Tb(3+) was bound to imprinted gel in shrunken state. This is because of the decrease of coordinated water molecules on Tb(3+) and the strong binding of this ion to methacrylate pairs which are encoded within the weakly cross-linked network of imprinted gel.     

Purification of muscle enzymes by pseudoaffinity chromatography

A method based on pseudoaffinity chromatography has been developed for the separation of lactate dehydrogenase (LDH), pyruvate kinase (PK) and aldolase from rabbit muscle extract using cross-linked guar (CLG) and cross-linked pectin (CLP) as the matrices, and dyes as the ligands. Screening of several dyes revealed that dyes No. 1014 and No. 1015, immobilized on CLG and CLP displayed a higher affinity for LDH and PK. Aldolase was not retained on any of the dye columns. It was observed that 1014-CLP and 1014-CLG columns retained 90% and 55% LDH activities, respectively, whereas 1015-CLP and 1015-CLG retained 83% LDH and 72% PK. A coupled-column system comprising 1014-CLP and 1015-CLP or 1014-CLG and 1015-CLG could separate LDH, PK, and aldolase from a mixture of these enzymes, as well as from rabbit muscle extract. Enzymes were found to be homogeneous on polyacrylamide gel electrophoresis. The method has been found to be simple and economical.     

Mechanism of inhibition of sickling by dimethyl adipimidate. Effects of intertetramer cross-linking

Dimethyl adipimidate (DMA), an effective antisickling agent in vitro, reacts with free amino groups producing chemically modified and cross-linked molecules. In this report, we have investigated the effect of cross-linked hemoglobin tetramers on sickle hemoglobin polymerization. Since the extent of cross-linking is pH-dependent, we first compared the solubilities of deoxygenated hemolysates prepared from sickle cells previously treated with dimethyl adipimidate at either pH 7.4 or 8.4. The solubility of the hemolysate increased from 18.6 +/- 0.8 g/dl in the untreated sample to 20.9 +/- 1.5 g/dl (pH 7.4) and to 25.4 +/- 3.0 g/dl (pH 8.4) after dimethyl adipimidate treatment. Removal of cross-linked hemoglobin tetramers from hemolysate obtained from dimethyl adipimidate-treated cells abolished part of this effect; at pH 7.4, the solubility decreased from 20.9 +/- 1.5 to 19.4 +/- 0.2 and at pH 8.4 from 25.4 +/- 3.0 to 21.0 +/- 1.5. However, the ratio of [14C]DMA-labelled hemoglobin in the sol phase to that in the gel phase in the unfractionated hemolysate was 1.17 at pH 7.4 and 1.25 at pH 8.4, suggesting that part of the cross-linked hemoglobin tetramers was incorporated into the gel. In order to further investigate the effect of cross-linked hemoglobin tetramers on sickle hemoglobin polymerization, we separated cross-linked hemoglobin tetramers on a gel-filtration column, prepared mixtures of untreated sickle hemoglobin and cross-linked hemoglobin tetramers and studied the polymerization of these mixtures. The Csat of the untreated hemolysate increased progressively from 18.6 +/- 0.8 to 22.5 +/- 0.8 g/dl with 33% cross-linked hemoglobin tetramers. The hemoglobin concentration in the gel decreased from 43 +/- 1.0 to 33.8 +/- 1.0 g/dl with 33% cross-linked hemoglobin tetramers, while the pellet volume fraction, phi p, increased with and almost approached 1 at 50% cross-linked hemoglobin tetramers. In addition, the sol phase contained a higher molecular weight distribution of cross-linked hemoglobin tetramers than the gel phase. These observations suggest that a loose polymer was formed in the gel phase with a hemoglobin concentration much lower than that of the control. Thus, polymerization of sickle hemoglobin is inhibited by: (1) exclusion of higher molecular weight cross-linked hemoglobin tetramers from the gel, and (2) loose incorporation of cross-linked hemoglobin tetramers into the gel, perhaps preventing lateral packing and formation of tightly ordered fibers.     

Cross-linking studies of the cholesterol hydroxylation system from bovine adrenocortical mitochondria

Cytochrome P-450SCC and adrenodoxin were cross-linked with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. The sample containing 94% of cross-linked complex and 6% of free cytochrome P-450SCC was obtained after purification on cholate-Sepharose. Cytochrome P-450SCC in cross-linked complex completely preserves its high-spin form in the presence of Tween 20 or pregnenolone. Utilization of radioactively labelled adrenodoxin, chemical cleavage of cytochrome P-450SCC from cross-linked complex with o-iodosobenzoic acid and HPLC for separation of peptides allow us to conclude that the complex of cytochrome P-450SCC with adrenodoxin was cross-linked through two amino acid sequences of cytochrome P-450SCC-Leu-88-Thr-107 and Leu-368-Gly-416. The cross-linked complex of adrenodoxin reductase, adrenodoxin and cytochrome P-450SCC with an apparent molecular mass of 114 kDa was obtained with N-succinimidyl-6-(4'-azido-2'-nitrophenylamino)hexanoate. The composition of cross-linked complex was determined by immunoblotting and by evaluation of radioactivity using preliminary N-ethyl[2,3-14C]maleimide-modified adrenodoxin. From this data it appears that the ternary complex may exist in solution.     

Evaluation of Integrin Molecules Involved in Substrate Adhesion

Integrins were cross-linked to their extracellular matrix ligands using non-penetrating chemical cross-linkers. This procedure did not disturb the distribution of integrin in the adhesion structure and adhesion plaque integrin staining remained even when the cultures were extracted with ionic detergents. 80-90% of the pi integrin in the cross-linked culture was extracted with RIPA buffer and the remaining 10-20% was recovered following reversal of the cross-linking. This separated two distinct integrin pools, one which can be cross-linked to substrate bound extracellular matrix and one which is not. The specificity of this procedure for cross-linking of integrins involved in substrate adhesion was demonstrated using NIH 3T3 cells which express both α5β1 and α5β1 integrins. α6 was cross-linked only in cells plated on laminin whereas α5 was cross-linked when fibronectin was present. Using antisera directed to the cytoplasmic domains of either α5 or β1 integrin, it was demonstrated that these domains can be blocked in the intact cell but the blocking can be removed using ionic detergent extraction after chemical cross-linking. The extracellular matrix associated with the substrate surface but not that associated with the media exposed surface is both cross-linked and retained on the plastic dish following cross-linking.     

Interaction of sugars, polysaccharides and cells with boronate-containing copolymers: from solution to polymer brushes

Interaction of mono- and disaccharides, polysaccharide particles and yeast cells with boronate-containing copolymers (BCC) of N-acryloyl-m-aminophenylboronic acid (NAAPBA) with N,N-dimethylacrylamide (DMAA) or N-isopropylacrylamide (NIPAM) was studied. The binding of saccharides to BCC of NIPAM resulted in a shift of its phase transition temperature (DeltaTP), which provided a quantitative measure for the complex formation. Among the sugars typical of non-reducing ends of glycoproteins the DeltaTP decreased in the order: N-acetylneuraminic acid > xylose approximately galactose > mannose approximately fucose > N-acetylglucosamine. Strong specific adsorption of the BCC on the cross-linked agarose gel Sepharose CL-6B (15-30 mg/ml gel at pH 9.2) was registered. The copolymers adsorption was due to boronate-sugar interactions and decreased with pH. Multivalent interaction of the BCC with the agarose gel has been proven by liquid column chromatography exhibiting a weak reversible adsorption of NAAPBA and almost irreversible adsorption of DMAA-NAAPBA copolymer from 0.1 M sodium phosphate buffer, pH 7.9. The two studied BCCs could be completely desorbed from the gel by 0.1 M fructose in aqueous buffered media with pH from 7.5 to 9.2. In turn, the agarose particles and yeast cells were found to adhere to siliceous supports end-grafted with boronate-BCC of N,N-dimethylacrylamide at pH > or = 7.5, due to the actions. Quantitative detachment of adhered particles or cells could be attained by addition of 20 mM or 100 mM fructose, respectively, in the pH range from 7.5 to 9.2. Affinity adhesion of micron-size carbohydrate particles to boronate-containing polymer brushes fixed on solid supports was considered as a model system suggesting a new approach to isolation and separation of living cells.     

Spray-dried chitosan microspheres containing 8-hydroxyquinoline -5 sulphonic acid as a new adsorbent for Cd(II) and Zn(II) ions

In the present study, a new chelating adsorbent was prepared from chitosan microspheres cross-linked with glutaraldehyde by spray drying using 8-hydroxyquinoline -5 sulphonic acid as chelant agent (CTS-SX-CL). Microspheres of the new adsorbent were characterized by Raman spectroscopy, scanning electron microscopy (SEM) and energy-dispersive X-ray microanalysis (EDX). The effect of pH, contact time and concentration of metallic ions in solution were evaluated on the adsorption behavior of Cd(II) and Zn(II) by CTS-SX-CL. Adsorption was maximum for both Cd(II) and Zn(II) at pH 8.0. Adsorption kinetic curves were obtained and could be fit by the pseudo second-order adsorption model. An analysis of equilibrium adsorption data using the Langmuir isotherm model indicated that the maximum adsorption capacity of CTS-SX-CL was higher than that of CTS-CL for both ions investigated. The adsorption capacity increased 74% for Cd(II).     

In vitro release of lysozyme from gelatin microspheres: effect of cross-linking agents and thermoreversible gel as suspending medium

This study was aimed to characterize the microstructure and the performance of gelatin microspheres (GMs) cross-linked by two different cross-linkers viz. d-glucose and glutaraldehyde. New formulations were obtained, suspending the GMs in a thermoreversible Pluronic F127 (PF127) liquid-crystalline gel. Lysozyme was used as a model biomacromolecular drug to evaluate release features. Both types of cross-linked GMs were prepared by thermal gelation method. The lysozyme-loaded microspheres were characterized by scanning electron microscopy (SEM) for size distribution, shape, and surface texture. SEM revealed that both types of lysozyme-loaded GMs were spherical in shape and that the surface of glutaraldehyde cross-linked GMs was smoother than that of the glucose cross-linked GMs. The degree of cross-linking of microspheres was investigated using ATR-FTIR technique. The prepared GMs were suspended in 20% w/v aqueous PF127 gel for which the usual sol-gel transition temperature of 22 °C did not change in the presence of GMs, as indicated by rheological measurements. SAXS study of the PF127 gel confirmed the occurrence of a discrete cubic liquid-crystalline phase of the Fm3m type whose lattice parameter slightly decreased as a result of GMs addition. The in vitro release of lysozyme from both types of cross-linked GMs was successfully controlled when they were suspended in PF127 gel, thus suggesting the potential use of this new combined formulation as a drug-depot system.     

Chemical cross-linking of chick oviduct progesterone-receptor subunits by using a reversible bifunctional cross-linking agent.

Chick oviduct progesterone-receptor proteins were treated in cytosol with the reversible cross-linking reagent methyl 4-mercaptobutyrimidate. The product of the reaction was a 7S complex that could be detected and recovered after sucrose-density-gradient centrifugation in 0.3M-KCl. The extent of the reaction was dependent on the concentration of methyl 4-mercaptobutyrimidate and independent of the presence of bound hormone, since unlabelled receptors could also be cross-linked. The cross-linking reaction required conditions in which the cytosol 6S complex was preserved. A Stokes radius of 7.3 nm was determined by gel filtration in Agarose A-1.5 m in 0.3 M-KCl. The sedimentation coefficient, which was also determined in 0.3 M-KCl, allowed us to calculate a mol. wt. of 228,000. We were also able to cross-link partially purified receptor forms isolated by using an Agarose A-15 m column. On reduction with beta-mercaptoethanol the complex broke down to 4S monomers that were identified by DEAE-cellulose and phosphocellulose chromatography, adsorption on DNA-cellulose and gel filtration in an Agarose A-1.5 m column. In most cases, A and B receptor proteins were released in equivalent amounts, implying that the cross-linked form was an A-B complex.     

Interaction of L-glutamate oxidase with triazine dyes: selection of ligands for affinity chromatography

Glutamate oxidase (GOX, EC 1.4.3.11) from Streptomyces catalyses the oxidation of L-glutamate to alpha-ketoglutarate. Its kinetic constants for L-glutamate were measured equal to 2 mM for Km and 85.8 s(-1) for kcat. BLAST search and amino acid sequence alignments revealed low homology to other L-amino acid oxidases (18-38%). Threading methodology, homology modeling and CASTp analysis resulted in certain conclusions concerning the structure of catalytic alpha-subunit and led to the prediction of a binding pocket that provides favorable conditions of accommodating negatively charged aromatic ligands, such as sulphonated triazine dyes. Eleven commercial textile dyes and four biomimetic dyes or minodyes, bearing a ketocarboxylated-structure as their terminal biomimetic moiety, immobilized on cross-linked agarose gel. The resulted mini-library of affinity adsorbents was screened for binding and eluting L-glutamate oxidase activity. All but Cibacron Blue 3GA (CB3GA) affinity adsorbents were able to bind GOX at pH 5.6. One immobilized minodye-ligand, bearing as its terminal biomimetic moiety p-aminobenzyloxanylic acid (BM1), displayed the higher affinity for GOX. Kinetic inhibition studies showed that BM1 inhibits GOX in a non-competitive manner with a Ki of 10.5 microM, indicating that the dye-enzyme interaction does not involve the substrate-binding site. Adsorption equilibrium data, obtained from a batch system with BM1 adsorbent, corresponded well to the Freundlich isotherm with a rate constant k of 2.7 mg(1/2)ml(1/2)/g and Freundlich isotherm exponent n of 1. The interaction of GOX with the BM1 adsorbent was further studied with regards to adsorption and elution conditions. The results obtained were exploited in the development of a facile purification protocol for GOX, which led to 335-fold purification in a single step with high enzyme recovery (95%). The present purification procedure is the most efficient reported so far for L-glutamate oxidase.     

Interactions between murine B lymphocyte surface membrane molecules. Loaded but not free receptors for complement and the Fc portion of IgG co-cap independently with cross-linked surface Ig

We have investigated the possible physical interactions between CR, receptors for the Fc gamma R and surface Ig (sIg) on the surface membrane of murine B lymphocytes. We used the rat mAb to murine CR, 8C12, and 7G6, as CR ligands, and soluble Ag-antibody complexes as FcR ligands; and F(ab')2 fragments of rabbit antibodies specific for mouse IgM and IgD as sIg ligands. We have found that: 1) sIg, CR, and Fc gamma R are not directly linked, because capping of any one did not affect the expression of the others; 2) the mAb 8C12 and 7G6 failed by themselves to cross-link CR; 3) soluble Ag-antibody complexes crosslinked some, Fc gamma R on a minority of Fc gamma R+ lymphocytes; 4) once loaded with anti-CR mAb, CR co-capped with sIg when sIg was cross-linked; 5) once loaded with Ag-antibody complexes, Fc gamma R also co-capped with sIg when sIg was sIg was cross-linked; 6) loading of Fc gamma R did not affect the co-capping of surface CR with cross-linked sIg and conversely, loading of CR did not affect the co-capping of Fc gamma R with cross-linked sIg; only loaded CR or Fc gamma R co-capped with sIg regardless of the status of the other surface molecule; 7) neither loaded nor free CR co-capped with cross-linked Fc gamma R, and neither loaded nor free Fc gamma R co-capped with cross-linked CR. These results demonstrate that both Fc gamma R and CR independently become associated with sIg when either receptor is loaded and sIg is cross-linked.     

Proteins of small subunits of rat liver ribosomes that interact with poly(U). II. Cross-links between poly(U) and ribosomal proteins in 40 S subunits induced by UV irradiation

(1) When rat liver 40 S ribosomal proteins in 6 M urea were were mixed with poly(U) at an appropriate ratio, a precipitate was formed which was also insoluble in the sample solution for two-dimensional acrylamide gel electrophoresis. Analyses by two-dimensional acrylamide gel electrophoresis showed that S7 and S10 proteins (according to our numbering system) had disappeared selectively from the fraction soluble in 6 M urea. These two proteins were present in the fraction insoluble in 6 M urea, and became soluble in the sample solution after treating it with RNase. The results suggest that S7 and S10 proteins have strong affinities for poly(U). When rat liver 40 S subunits were incubated with poly(U), similar results were obtained. (2) After incubation of 40 S subunits with [3H]poly(U) and then with unlabeled poly(U), UV irradiation cross-linked poly(U) to the protein moiety of the 40 S subunit. When the protein fraction insoluble in the sample solution for two-dimensional electrophoresis was prepared from 40 S subunits cross-linked to poly(U) and then subjected to two-dimensional acrylamide gel electrophoresis after RNase treatment, S7 and S10 proteins were detected on the gel. In addition to the S7 protein spot, a triangular area spreading from the spot to the origin contained radioactivity. The results suggest that poly(U) is cross-linked to S7 protein and oligo(U) fragments bound to S7 protein affect its electrophoretic mobility. (3) Ribosomal proteins were prepared from 40 S subunits cross-linked to carrier-free [3H]poly(U) and analyzed by three-dimensional acrylamide gel electrophoresis (Terao, K. & Ogata, K. (1975) Biochim. Biophys. Acta 402, 214--229) after RNase treatment. It was found that S7, S6, and S15 proteins are cross-linked to poly(U). From the results of the present and preceding experiments it is concluded that S7 is the poly(U)-binding protein. The possibility that other proteins in 40 S ribosomal subunits interact with poly(U) is discussed.     

Syntheses of Isoquinolines by Condensation of Dopamine with Carbonyl Compounds

A cleavable photoactivable heterobifunctional reagent, the N-hydroxysuccinimide ester of 1- azido-5-naphthalene sulfonyl S-carboxymethylthiocysteamine (NHS-ANS-CTC), was synthesized and characterized. The reagent was applicable to the group-directed modification of protein ligands, such as an invertebrate lectin and a trypsin inhibitor. The modified ligands bound to rabbit erythrocyte ghosts and trypsin, respectively. Upon exposure to ultraviolet light, the modified ligands reacted with their binding proteins to form cross-linked fluorescent products. The cross-linked fluorescent complexes were readily cleaved by reducing the disulfide bond of the reagent, leaving the fluorescent probe on the binding proteins. The photolabeled binding proteins were analyzed by SDS-polyacrylamide gel electrophoresis. The fluorescence intensity of the fluorescent probe was enhanced by 4~8 times to improve sensitivity when the SDS-gel was dehydrated with methanol. This phenomenon was also observed with the proteins labeled with 1-dimethylamino-5-naphthalene sulfonyl chloride.     

Preparation and characterization of silica gel/chitosan composite for the removal of Cu(II) and Pb(II)

Silica gel/chitosan composite (SiCS) was prepared via., sol-gel method by mixing silica gel and chitosan and cross-linked with bifunctional cross-linker glutaraldhyde. The SiCS composite was characterized using FT-IR, SEM-EDAX, XRD and BET methods. The sorption of copper and lead ions onto SiCS has been investigated. The SiCS composite was found to have excellent metal sorption capacity than the silica gel (Si) and chitosan (CS). The sorption experiments were carried out in batch mode to optimize various parameters viz., contact time, pH, initial metal ion concentration, co-ions and temperature that influence the sorption. Langmuir, Freundlich and Dubinin-Radushkevich adsorption isotherm models were applied to describe isotherm constants. Equilibrium data agreed well with the Freundlich isotherm model. Thermodynamic studies revealed that the nature of sorption is spontaneous and endothermic. The SiCS removes metals by means of adsorption and complexation. Sorption capacity of SiCS is compared with other sorbents which suggest that this composite was useful for removing copper and lead from aqueous solution.     

Protein interaction with immobilized ligands: quantitative analyses of equilibrium partition data and comparison with analytical chromatographic approaches using immobilized metal affinity adsorbents

Quantitative or analytical affinity chromatography has been successful primarily for the analysis of biologically determined macromolecular affinity relationships. Quantitative approaches are also needed to better characterize simpler, chemically defined immobilized ligands with potential for selective interaction with specific, predetermined protein surface groups. Protein interaction with immobilized metal is a rather selective and versatile, high-affinity adsorption technique for which there is little quantitative information. Using model protein interactions with immobilized Cu2+ ions, we have compared analytical frontal affinity chromatographic methods to a simple, nonchromatographic protocol for the rapid determination of quantitative affinity relationships. Values obtained for the equilibrium dissociation constant (Kd) and binding capacity (Lt) characterizing the interaction of lysozyme with immobilized Cu2+ were quite similar by frontal analysis (Kd = 37-42 X 10(-6) M; Lt = 6.8-7.4 X 10(-6) mol protein/ml gel) and by equilibrium binding analyses (Kd = 33 +/- 4.7 X 10(-6) M; Lt = 5.8-6.1 X 10(-6) mol protein/ml gel; 14 determinations). The interaction of ovalbumin with immobilized Cu2+ was characterized by an affinity (Kd = 4.2-4.8 X 10(-6) M) and capacity (Lt = 1.5-2.1 X 10(-6) mol protein/ml gel) which were also the same regardless of the method for affinity analysis. These values indicate that the total protein bound at saturation corresponds to as much as 17% of the total immobilized Cu2+ ions (approximately 40 X 10(-6) mol/ml gel). Thus, depending on the fraction of total immobilized Cu2+ available for interaction with a given protein (e.g., lysozyme), the number of individual immobilized ligands actively participating as well as those rendered unavailable upon individual protein binding events may be greater than 1. Linear Scatchard plots obtained for both lysozyme and ovalbumin (purified) suggest the presence of only a single type of immobilized Cu2+-protein interaction operative under the experimental conditions employed. However, Scatchard analyses of data obtained by the nonchromatographic equilibrium binding method also demonstrated the ability to simultaneously resolve the contribution of two components whose presence was predicted by frontal chromatography. Our results support the validity and utility of equilibrium binding data analyzed according to the equations outlined by Scatchard and others as an alternative to analytical chromatographic methods.     

Immobilization of Cross-Linked Phenylalanine Ammonia Lyase Aggregates in Microporous Silica Gel

A separable and highly-stable enzyme system was developed by adsorption of phenylalanine ammonia lyase (PAL) from Rhodotorula glutinis in amino-functionalized macroporous silica gel and subsequent enzyme crosslinking. This resulted in the formation of cross-linked enzyme aggregates (PAL-CLEAs) into macroporous silica gel (MSG-CLEAs). The effect of adsorptive conditions, type of aggregating agent, its concentration as well as that of cross-linking agent was studied. MSG-CLEAs production was most effective using ammonium sulfate (40%-saturation), followed by cross-linking for 1 h with 1.5% (v/v) glutaraldehyde. The resulting MSG-CLEAs extended the optimal temperature and pH range compared to free PAL and PAL-CLEAs. Moreover, MSG-CLEAs exhibited the excellent stability of the enzyme against various deactivating conditions such as temperature and denaturants, and showed higher storage stability compared to the free PAL and the conventional PAL-CLEAs. Such as, after 6 h incubation at 60°C, the MSG-CLEAs still retained more than 47% of the initial activity whereas PAL-CLEAs only retained 7% of the initial activity. Especially, the MSG-CLEAs exhibited good reusability due to its suitable size and active properties. These results indicated that PAL-CLEAs on MSG might be used as a feasible and efficient solution for improving properties of immobilized enzyme in industrial application.     

Increasing immunoglobulin G adsorption in dextran‐grafted protein A gels

The formation of a stable spatial arrangement of protein A ligands is a great challenge for the development of high‐capacity polymer‐grafted protein A adsorbents due to the complexity in interplay between coupled ligands and polymer chain. In this work, carboxymethyl dextrans (CMDs) with different molecular weight were introduced to provide stable spatial ligand arrangement in CMD‐grafted protein A gels to improve IgG adsorption. The result showed that coupling of protein A ligand in CMD‐grafted layer had no marked influence on pore size and dextran layers coupled with the ligands were stable in experimental range of salt concentrations. The result of IgG adsorption revealed that carboxymethyl dextran T10, a short CMD, was more suitable as a scaffold for the synthesis of high‐capacity protein A gels. Moreover, the maximal adsorption capacity for IgG was obtained to be 96.4 mg/g gel at ionic capacities of 300–350 mmol/L and a ligand density of 15.2 mg/g gel. Dynamic binding capacity for IgG exhibited a higher capacity utilization in CMD‐grafted protein A gels than non‐grafted protein A gel. The research presented a tactics to establish a stable dextran layer coupled with protein A ligands and demonstrated its importance to improve binding capacity for IgG.