The influence of bond chemistry on immobilized enzyme systems for ex vivo use

The ideal derivatized support for the clinical use of an immobilized enzyme system should irreversibly bind active enzyme. We have investigated the behavior of heparinase and bilirubin oxidase immobilized via cyanogen bromide, tresyl chloride, epoxide, or carbodiimidazole activated natural and synthetic matrices. The protein bound to each activated support was 90% for cyanogen bromide (CNBr) activated agarose, 50-80% for tresyl chloride activated agarose, and 50% for oxirane activated acrylic (Eupergit C). The activity retention of immobilized heparinase was greatest (50%) with CNBr activated agarose while for the immobilization of bilirubin oxidase, the activity retention was greatest (25-30%) with tresyl chloride activated agarose and oxirane activated acrylic.The stability of the different covalent bonds was studied in vitro with radioiodinated enzymes. The leaching profiles showed the same trends for each support and chemistry. A plateau in portein leaching was reached after a few hours of incubatttion and the transient leaching period was well represented byu a logarithimic function of time. The amount of enzyme released from the least stable support (CNBr activated agarose) in 24 h was injected intravenously in New Zealand white rabbits. Using an indirect enzyme-linked immunnosorbant assay (ELISA), no immune responce was detected. The transient leaching profile was shortenend by washingthe enzyme-support conjugate with 1M hydroxylamine, pH8.5 intermolecular cross-linking with glutaraldehyde also improves the enzyme-support stability. Tresyl chloride and oxirane activated supports produce bonds with improved stability without adversely affecting enzymatic activity.     

Stability of immobilized soybean lipoxygenases: influence of coupling conditions on the ionization state of the active site Fe

The potential application of lipoxygenase as a versatile biocatalyst in enzyme technology is limited by its poor stability. Two types of soybean lipoxygenases, lipoxygenase-1 and -2 (LOX-1 and LOX-2) were purified by a two step anion exchange chromatography. Four different commercially available supports: CNBr Sepharose 4B, Fractogel((R)) EMD Azlactone, Fractogel((R)) EMD Epoxy, and Eupergit((R)) C were tested for immobilization and stabilization of the purified isoenzymes. Both isoenzymes gave good yields in enzyme activity and good stability after immobilization on CNBr Sepharose 4B and Fractogel((R)) EMD Azlactone. Rapid decay in activity associated with change in the ionization state of Fe, as shown by EPR measurements was observed within the first 5 days after immobilization on epoxy activated supports (Eupergit((R)) C and Fractogel((R)) EMD Epoxy) in high ionic strength buffers. Stabilization of the biocatalyst on these supports was achieved by careful adjustment of the immobilization conditions. When immobilized in phosphate buffer of pH 7.5 and low ionic strength (0.05 M), the half-life time of the immobilized enzyme increased 20 fold. The dependence of the stability of LOX immobilized on epoxy activated supports on the coupling conditions was attributed to a modulation of the ligand environment of the iron in the active site and consequently its reactivity.     

Preparation of immobilized NAD glycohydrolase from Neurospora crassa conidia by hydrophobic interaction--characteristics of the enzyme derivative

NAD glycohydrolase from Neurospora crassa conidia has been immobilized by hydrophobic interaction on Sepharose 4B beads coated with propyl residues through CNBr activation. The bond resulted stable under a wide range of conditions (ionic strength, temperature, pH). As a result of immobilization the pH optimum for catalytic activity shifted by about 0.2 pH unit in the acidic direction, to lie between 7.5 and 7.3. The stability of the enzymatic activity was largely enhanced by effect of immobilization but the Km value towards NAD+ was increased compared with that of the free enzyme (1 X 10(-3) and 2 X 10(-4) M respectively).     

Amino acid ester synthesis by immobilized α-chymotrypsin

Summary A screening of immobilized -chymotrypsin preparations suitable for the synthesis of N-acetyl-L-tyrosine ethyl ester from N-acetyl-L-tyrosine and up to 99% ethanol was carried out. -Chymotrypsin adsorbed to Sepharose LH-20 or covalently bound to Sepharose 4B (tresyl chloride activation) was found to be an efficient catalyst. A column packed with immobilized enzyme retained 60% of its initial activity after 6 days of operation in a cyclohexane-ethanol medium.     

Purification of chymotrypsin by subunit exchange chromatography on the denatured protein

The alpha-chymotrypsin subunits immobilized under denaturing conditions (6 M urea or 1% SDS) on CNBr-activated Sepharose 4B, were found to interact with soluble chymotrypsin subunits with the formation of oligomers higher than dimers. Subunits immobilized under nondenaturing conditions form only dimers. The effects of several parameters, such as organic solvents, cations, and anions of the lyotropic series, on the associating properties of the immobilized derivatives were examined. The interaction between immobilized and free enzyme was shown to be specific because extraneous proteins and compounds were not bound by the derivatives. Chymotrypsinogen, studied analogously, did not show appreciable self-associating capacity. Chymotrypsin subunits immobilized under denaturing conditions and packed in a column proved to be suitable for the purification of chymotrypsin from both bovine and porcine pancreatic extracts. The "subunit exchange" chromatography of such extracts, carried out between pH 2.5 and 4, gave an eightfold purification with a 93% recovery of chymotryptic activity. The specific activity was ca. 12,000 Schwert and Takenaka units/mg. Only 6% of the tryptic activity was bound by the column. The capacity of the matrix, 6 mg chymotrypsin/mL, dropped to about 70% of the original value after.     

三乙醇胺基强碱性聚苯乙烯树脂共价固定胰酶的研究

用多孔强碱型三乙醇胺基聚苯乙烯阴离子交换树脂做为载体,用CNBr与载体上的多羟基作用共价偶联了胰酶。红外光谱表明:其共价偶联反应机理与用CNBr活化多糖类载体并接酶的机理相类似。最适偶联条件研究表明:CNBr用量增多,酶蛋白载量增加。但比活下降。偶联pH为10时,固定化酶有适宜的载量和较高的比活。由于胰酶水解蛋白反应释放出H~+质子,这些质子在载体内积累,使微环境内H~+质子浓度增加,进而使得固定化胰酶的pH—活性曲线在pH9～11范围内未出现下降。在变温和60℃恒温下对固定化酶的热稳定性测试表明:固相酶的热稳定性比天然酶的热稳定性有所提高。     

Peptide synthesis in aqueous-organic solvent mixtures with alpha-chymotrypsin immobilized to tresyl chloride-activated agarose

alpha-Chymotrypsin was immobilized with a high coupling yield (up to 80%) to tresyl chloride activated Sepharose CL-4B.The immobilized enzyme was tested for its ability to synthesize soluble peptides from N-acetylated amino acid esters as acyl donors and amino acid amides as acceptor amines in water-water-miscible organic solvent mixtures. It was found that the yield of peptide increased with increasing concentration of organic cosolvent. Almost complete synthesis (97%) of Ac-Phe-Ala-NH(2) was obtained from Ac-Phe-OMe using a sixfold excess of Ala-NH(2). The rate of peptide formation in aqueous-organic solvent mixtures was good. Thus, 0.1M peptide was formed in less than 2 h in 50 vol% DMF with 0.1 mg immobilized chymotrypsin/mL reaction mixture. The immobilized enzyme distinguished between the L and D configurations of acceptor amino acid amides even in high concentration of nonaqueous component (90% 1,4-butanediol). The effect of temperature was studied. It was found that both the yield of peptide and the stability of immobilized enzyme increased when the temperature was lowered. Experiments could be performed at subzero temperatures in the aqueous-organic solvent mixtures resulting in very high yield of peptide. After three weeks continuous operation at 4 degrees C in 50% DMF, the immobilized enzyme retained 66%of its original synthetic activity. The activity of the immobilized enzyme was better conserved with a preparation made from agarose with a higher tresyl group content compared to a preparation made from a lower activated agarose, indicating that multiple point of attachment has a favorable effect on the stability of the enzyme in aqueous-organic solvent mixtures. The major advantage of using water-miscible instead of water-immiscible organic solvents to promote peptide syntheses appears to be the increased solubility of substrates and products, making continuous operation possible.     

Use of epoxysepharose for protein immobilisation

Epoxy Sepharose, an activated affinity matrix which has been used for immobilisation of carbohydrates has been tried for immobilisation of proteins. Under normal conditions of coupling at neutral or alkaline pH proteins do not couple to epoxy Sepharose. However, a very high salt concentration during coupling allows the binding of proteins to epoxy Sepharose at a pH as low as 8.5. Increasing ionic strength and/or pH facilitates the binding. The bioactivity of the proteins is not destroyed by the immobilisation. This matrix, unlike cyanogen bromide-Sepharose, retains its ability to bind albumin by 80–90% even after 60 days of storage in aqueous suspension at 4°C. Its capacity to bind proteins is comparable to that of cyanogen bromide-Sepharose.     

Tresyl activation of a hydroxyalkyl ligand for coupling to a hydrazide gel: stable immobilization of T-2 toxin for affinity purification of T-2 antibody

A stable T-2 hydrazide gel is prepared by activating T-2 toxin with tresyl chloride followed by coupling to agarose-adipic acid hydrazide. Utilized as an affinity chromatography column, this T-2 hydrazide gel purifies a monoclonal antibody for T-2 in high yield directly from ascites fluid. Specific antibody trapped on the column is eluted either with excess T-2 or at pH 11.6. Much less successful are two other T-2 affinity columns that were prepared and evaluated: T-2 bovine serum albumin Affi-Gel 15 and T-2 hexylamine Sepharose.     

Immobilized human hemoglobin,a versatile matrix for analytical and biotechnological applications

The analytical and biotechnological applications of human hemoglobin immobilized covalently on CNBr–Sepharose 4B are reviewed. Hemoglobin is bound to the matrix as αβ dimers via either chain. The immobilized αβ dimers maintain the capacity to interact reversibly with soluble ones under conditions where the soluble protein is in self-association equilibrium. Under these conditions, therefore, immobilized dimers bind part of the soluble protein. In turn, the binding process can be used to assess the specific features of the equilibrium on solid-phase and to extract selectively hemoglobin from a variety of biological specimens of practical interest. A different application of immobilized αβ dimers concerns their use in the determination of the equilibrium and kinetic stability of the heme–globin linkage, a property that is directly correlated with the stability of the hemoglobin molecule. The advantages and limitations attendant the use of the immobilized protein relative to the soluble one are discussed.     

Covalent immobilization of unilamellar liposomes in gel beads for chromatography

For immobilized (proteo)liposome chromatography, unilamellar liposomes were covalently bound within gel beads that had been activated by CNBr, N-hydroxysuccinimide, tresyl, or chloroformate. Liposomes composed of phosphatidylcholine (PC) and 2 mol% of amino-containing lipid (phosphatidylethanolamine-caproylamine) were immobilized in the activated gels at 5-35 micromol lipid/ml gel and yields of 11-70%. The highest immobilized amount was found in chloroformate-activated TSK G6000PW gel, which contains large pore size (>100 nm). Liposomes composed of PC alone could also be attached to the chloroformate-activated gels at 33-42 micromol/ml gel and yields of 58-65%, probably by crosslinking of the phosphate moiety of phospholipid with the active group of the adsorbent. Liposomes prepared by various phospholipids with or without amino-containing lipids can generally be immobilized in the chloroformate-activated gels. The covalently bound liposomes were characterized by their high stability, unilamellarity, permeability of the membranes, and drug-membrane partition properties. A stable membrane phase was constructed for chromatographic experiments to be performed under extreme elution conditions.     

The properties of subunits of avidin coupled to Sepharose

Avidin that had been coupled to Sepharose 4B activated with CNBr retained over 90% of its biotin-binding capacity. When low concentrations of CNBr were used about 75% of the protein could be removed from the Sepharose by washing with guanidinium chloride (6 m). The remaining 25%, the covalently bound subunits, had an almost undiminished capacity for biotin but a decreased affinity. Addition of avidin subunits in guanidinium chloride to the coupled subunits followed by dilution or dialysis restored the original biotin-binding capacity and affinity. Three classes of binding sites were present in preparations of the subunits. About 25% were weak (K=5x10(-8)m), about one third exchanged their biotin in a few minutes (K approximately 10(-10)m) and the remainder were indistinguishable from the native tetramer. The last-named exchanged their bound biotin at a similar rate at pH5 and at pH2, they did not lose their biotin in 6 m-guanidinium chloride and they were resistant to tryptic digestion in the absence of biotin. The proportion of these stable sites could be increased to 65% when the subunits coupled to Sepharose were incubated at 37 degrees C. This increase was reversed by guanidinium chloride, which suggested that it was caused by a temperature-dependent association of covalently linked subunits. This in turn implies a temperature-dependent mobility of the agarose matrix of the Sepharose. Analysis of the spatial distribution of subunits within the Sepharose beads led to the conclusion that the association of subunits implied that they could move through distances greater than 20nm (several hundred A). This mobility and consequent formation of tetramer was greatly decreased when avidin subunits were coupled to Sepharose that had been cross-linked with divinyl sulphone.     

Rapid isolation of human plasma anti-alpha-galactoside antibody using sugar-specific binding to guar galactomannan or agarose.

A method of purifying the naturally occurring antibody to alpha-galactoside moiety (anti-alpha-Gal) in human plasma by a single-step affinity chromatography on cross-linked guar galactomannan (CLGG) or agarose (Sepharose 4B) is described. IgG nature of the two preparations, as revealed by agar gel diffusion, as well as their preference for alpha-anomer of galactose, as revealed in inhibition of their agglutination of trypsinized rabbit erythrocytes by sugars, identified them with anti-alpha-Gal. The antibody binding capacity of Sepharose 4B was only a third of that of CLGG. Both gels showed similar dependence on ionic strength for binding. The pH optimum for binding of anti-alpha-Gal to CLGG was 8.0. Significantly anti-alpha-Gal binding to Sepharose was unaffected by CNBr activation and ligand coupling to the gel, thus warning that contaminating plasma could introduce artifacts in agarose-based chromatography of human tissue biomolecules.     

Protein A immobilized polyhydroxyethylmethacrylate beads for affinity sorption of human immunoglobulin G

Protein A immobilized polyhydroxylmethyacrylate (PHEMA) microbeads were investigated for the specific removal of HIgG from aqueous solutions and from human plasma. PHEMA microbeads were prepared by a suspension polymerization technique and activated by CNBr in an alkaline medium (pH 11.5). Protein A was then immobilized by covalent binding onto these microbeads. The amount of immobilized protein A was controlled by changing pH and the initial concentrations of CNBr and protein A. The maximum protein A immobilization was observed at pH 9.5. Up to 3.5 mg protein A/g PHEMA was immobilized on the CNBr activated PHEMA microbeads. The maximum HIgG adsorption on the protein A immobilized PHEMA microbeads was observed at pH 8.0. The non-specific HIgG adsorption onto the plain PHEMA microbeads was low (about 0.167 mg of HIgG/g PHEMA). Higher adsorption values (up to 6.0 mg of HIgG/g PHEMA) were obtained in which the protein A immobilized PHEMA microbeads were used. Much higher amounts of HIgG (up to 24.0 mg of HIgG/g PHEMA) were adsorbed from human plasma.     

Factors affecting the properties of insolubilized anitbodies.

IgG separated from an antiserum to estradiol was coupled under various experimental conditions to Sepharose activated either with CNBr or by conversion into a long-armed derivative (the N-hydroxysuccinimide ester). The conjugates were characterized by measurement of the binding parameters, in order to evaluate separately the loss of sites and the loss of affinity. The cross-reactivity with estriol and estrone was measured to obtain information on the occurrence of structural alterations of the antibody site. The results show that the loss of immunoreactivity varies in extent (from 95% to less than 10%) and in nature (loss of sites or of affinity or a combination of both effects) depending on the coupling conditions. The use of a hydrocarbon extension to keep the protein distant from the matrix does not prevent the loss of active sites but is effective in safeguarding the affinity of the residual sites. The loss of sites can be substantially reduced by coupling at a pH value around neutrality and by keeping the protein/matrix mass ratio low. At a coupling pH of 6.4 and at a mass ratio of 0.1-0.2 nmol IgG/mg of Sepharose, the antibodies were insolubilized with a negligible loss of sites and affinity; on increasing the mass ratio (up to 10 nmol IgG/mg Sepharose) there is a progressive loss of sites accompanied by a substantial lowering of the affinity of the residual sites. On the basis of the above-mentioned findings, the nature of the effects occurring when antibodies are transferred from solution onto a solid matrix is discussed.     

Preparation of pyridoxal 5'-phosphate-bound sepharose and its use for immobilization of tryptophanase

Pyridoxal 5′-phosphate-bound Sepharose (SP) was prepared by coupling pyridoxal 5′-phosphate (PLP) to diazotized p-aminobenzamidohexyl-Sepharose. A derivative of pyridoxine having an absorption maximum at $\text{ca.}$ 316 nm (possibly, 6-amino-pyridoxine 5′-phosphate) was liberated from SP by treatment with 0.1 M sodium dithionite at pH 9.0. SP catalyzed the cleavage of tryptophan in the presence of Cu²⁺, a typical non-enzymatic model of tryptophanase reaction. From the spectrophotometric data and catalytic activity, it was estimated that SP contained about 1.5 μmoles of bound PLP per gram of Sepharose. Tetrameric apotryptophanase was immobilized by incubation with SP, followed by reduction with NaBH₄. The resulting immobilized tryptophanase retained $\text{ca.}$ 60 % of the catalytic activity of free tryptophanase used. This method was much superior to other methods used commonly for preparation of immobilized enzymes.     

Affinity chromatography of fibrinogen on Lens culinaris agglutinin immobilized on CNBr-activated sepharose: study of the active groups involved in nonspecific adsorption

Fibrinogen binds specifically to Lens culinaris agglutinin coupled to CNBr-activated Sepharose. However, a fraction of the retained fibrinogen remains tightly bound to the gel and is eluted only by electrophoretic desorption. The irreversible binding of fibrinogen results from the interaction of fibrinogen specifically bound to the immobilized lectin with some reactive groups still present on the Sepharose matrix. Therefore, the active groups present on lectin-Sepharose after different treatments and their influence on the irreversible binding of fibrinogen have been studied. After the coupling step some cyanate esters remain on the gel, but they are neutralized under all the conditions studied. In addition, imidocarbonates formed under the basic conditions used to activate Sepharose, and carbonates resulting from acid treatment of the gel, are also present. Carbonates seem to be the main active groups involved in the irreversible binding of fibrinogen to lectin-Sepharose. Imidocarbonates also contribute to the nonspecific binding, although to a lesser extent than carbonates. Treatment of CNBr-activated Sepharose with 0.1 M HCl prior to the coupling step and neutralization, after coupling, with 0.1 M ethanolamine, pH 9.5, for 24 h at room temperature reduce the nonspecific binding to less than 9% of the fibrinogen fraction retained by the column. This percentage is appreciably smaller than that obtained by neutralization for 2 h at room temperature with either 0.1 M Tris-HCl, pH 8.0 (congruent to 66%), or 1 M ethanolamine, pH 9.0 (congruent to 23%).     

Isopycnic-centrifugation studies in caesium chloride and in caesium sulphate on dermatan sulphate proteoglycans from bovine sclera

1. Frog epidermis tyrosinase was coupled to Sepharose activated with low concentrations of CNBr. The tetrameric form of the enzyme was linked to the matrix via its subunits. Dissociation of the bound active enzyme with guanidinium chloride yielded an active immobilized dimeric derivative. 2. Immobilized dimeric derivative was able to interact with soluble subunits formed transiently during renaturation. An 85% recovery of the native dopa oxidase specific activity was achieved after hybridization. 3. Fluorescence spectra of different immobilized derivatives suggested that tryptophan residues were involved in the interactions between tyrosinase subunits. 4. It is suggested that the activation of the subunits of tyrosinase involves a conformational change towards a more unfolded state, which favours a reassociation to the dimeric active state.     

Affinity chromatography of porcine pepsin and pepsinogen using immobilized ligands derived from the specific substrate for this enzyme

Affinity chromatography of porcine protease and its zymogen was carried out on immobilized components of specific substrate used for the pepsin determination. For the immobilization of N-acetyl-L-phenylalanine and iodinated derivative of L-tyrosine, divinyl sulfone activated Sepharose was used. Ligands with blocked amino group and free carboxyl one were linked to Sepharose via ethylene diamine spacer using carbodiimide reaction. Conditions of affinity chromatography of porcine pepsin and pepsinogen on the prepared carriers were optimized: the effect of pH, ionic strength and a nature of the buffers used on adsorption of the enzyme and zymogen to an affinity carrier, as well as their elution was studied. The following parameters were taken into consideration: capacity of the prepared affinity matrices, reproducibility of experiments and the enzyme stability. Pepsin was adsorbed to both immobilized ligands at pH 3.5-4.0; for the elution of the enzyme it was necessary to increase ionic strength (up to 0.5 M). For the adsorption of pepsinogen pH 5.2 was found to be optimum, for its desorption, an increase of ionic strength was used.