Isolation and purification of biopolymers by affinity chromatography. VI. Preparation and properties of an affinity adsorbent with a polysaccharide spacer for the purification of proteolytic enzymes

A new affinity sorbent has been synthesized — soybean trypsin inhibitor (STI)-amylopectin-hydrazidosuccinyl-Sepharose — and its properties have been studied in comparison with those of an analogous adsorbent without the spacer STI-Sepharose. The STI-amylopectin-hydrazidosuccinyl-Sepharose adsorbent has been used for the purification of trypsin from porcine pancreas and of callicrein from human blood plasma.     

Coupling of effector molecules to solid supports

In view of the limited stability of the isourea bond, formed in ligand coupling to CNBractivated polysaccharides, an alternative to this current activation method has been developed. 2,4,6-Trifluoro-5-chloropyrimidine (FCP), known as a reactive group in reactive dyes, was used to activate Sepharose. Under appropriate conditions a thermally stable product with unimpaired beaded structure was obtained, which was reactive toward amines and mercaptans. Coupling with hexamethylenediamine, aniline, and ethanethiol, respectively, yielded an incorporation of 0.2-2.7, 0.9-1.7, and 1.1 mmol ligand/g dry agarose. The stability of immobilized ligands based on FCP-Sepharose between pH 4 and 8 was about 200 times higher as compared to products originating from CNBr-Sepharose; ligand leakage was only 0.5 x 10^p-3%/h. The possibility of obtaining a high degree of substitution is a further advantage of the FCP activation. In addition, the FCP-activated Sepharose can be stored in the wet state at 4°C without substantial decrease in coupling capacity. The FCP analogs 2,4,5,6-tetrachloro- and 2,4,5,6-tetrafluoropyrimidine, and other polymers (cellulose, Sephadex, aminomethylpolystyrene) appeared to be applicable also.     

Analysis of the reaction kinetics of single esterase sepharose beads by microfluorometry of the fluorogenic substrate turnover

Heterogeneous catalysis of esterase (E.C. 3.1.1.1) immobilized on CNBr-Sepharose 4B was analyzed by microfluorometry. The hydrolysis of fluorescein diacetate was measured within single esterase-Sepharose beads during steady-state turnover. Fluorescence intensity profiles, total intensities, and reaction rates of single beads were measured by means of a microfluorometer. Their suitability for determining the kinetic constants of the system was evaluated by comparison with the theoretical values of the kinetic model. The computation was based on the kinetic equations describing the interaction of internal diffusion and enzymatic reaction with noncompetitive product inhibition within spherical particles. A method is described which can be used to derive the true K_m of the immobilized enzyme from a correlation of intensity- and turnover-dependent apparent K_m—s. Effectiveness factors and Damkohler numbers for individual esterase-Sepharose beads were determined from relative total fluorescence intensities and fluorescence intensity profiles, respectively.     

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.     

Ethylenediamine-Derived Chromatographic Ligand to Separate BSA,Lysozyme, and RNase A

Chromatography has become an essential tool for the purification of proteins, since most purification schemes involve some forms of this methodology. Recently, using chromatographic matrices prepared from symmetrical aminosquarylium cyanine dyes immobilized on Sepharose via a central alkylamino residue, we were able to isolate lysozyme, α-chymotrypsin and trypsin from a mixture. Following this, we envisioned that the immobilization of an asymmetric squarylium dye bearing an N-carboxyethyl group in one of its ending nuclei, on ethylenediamine-activated Sepharose, through EDC/NHS amidation coupling, could enhance the ligand’s mobility and improve the interactions with the target proteins. The prepared support was found to separate an artificial mixture of BSA, lysozyme, and RNase A. Unexpectedly, the support prepared in the absence of the dye exhibited a separation performance similar to that of the dyed support, contrary to that observed in all previous studies using cyanine dyes as ligands for affinity chromatography, which prompted us to try to determine the structural molecular constitution of the matrix surface. A synthetic route to the final chromatographic support could be devised, which is believed to consist in the cyclization of two nearby ethylenediamine units, involving the inclusion of a succinimide-derived residue between them and the EDC-mediated Lossen rearrangement of an intermediary hydroxamic acid.     

Preparation of immobilized sepharose-micrococcal nuclease derivatives: activity and stability

Micrococcal nuclease has been covalently attached to CNBr-activated Sepharose 4B by coupling through three different enzyme functions: (a) amino groups; (b) carboxyl groups; and (c) tyrosyl or histidyl residues. On the basis of coupling yield and catalytic efficiency, Sepharose-(NH₂) nuclease derivatives were chosen for further activity andstability studies. The activity of the insoluble enzyme has been evaluated with macromolecular (DNA) and small (synthetic nucleotide) substrates; with the latter the enzyme retains 70% of native enzyme activity. Good enhancement of enzyme stability in the 4–40°C range has been observed.     

Purification and some properties of tryptophan-5-monooxygenase from rabbit hindbrain

An affinity chromatography procedure for the rapid purification of tryptophan-5-monooxygenase from rabbit hindbrains was developed using e-aminocaproyl-D-tryptophan methyl ester-Sepharose-4B gels. The precise requirements for the optimal biospecific interaction between the affinity ligand and the ligate (enzyme) was established from a study of the effects of the variation in the length of the "spacer’’ on the affinity properties of the gel. The enzyme preparation isolated by this procedure was found to be essentially homogeneous and was characterized by a molecular weight of 200,000 ±20,000. SDS-polyacrylamide gel electrophoresis of the enzyme revealed it to be a dimer, the molecular weight of each subunit being approximately 90,000. The specific activity of the enzyme preparation is approxi-mately 7-10 times that of the crude homogenate, but a further fivefold enhancement in the specific activity could be obtained by limited proteolysis with trypsin. The extreme lability of the enzyme could be circumvented by its immobilization on activated Sepharose or by cross-linking with dimethyl suberimidate. The kinetic properties as well as the advantages of such stabilized enzyme preparations are presented.     

Properties of human parotid amylase immobilized by covalent binding to glass or sepharose or by reaction with immobilized antibody

Human parotid amylase was immobilized by covalent binding to CNBr-activated Sepharose, to Corning GAO-3940 silica glass biomaterial support by the diazonium reaction or reaction with glutaraldehyde, or as a result of the antigen-antibody reaction between rabbit antihuman parotid amylase IgG that was covalently bonded to GAO glass and soluble amylase. The amylase directly bonded to the supports showed constant activity at flow rates of 3-15 ml/min through a 1.76-cm³ (8-mm diameter) support bed, did not lose enzyme into a circulating starch solution, retained its activity in the presence of soluble antiamylase IgG, was optimally active at 35°-40°C, and lost activity at 40°-45°C. When the enzyme was bound by interaction with immobilized antibody, full activity was expressed, but some enzyme was solubilized by a circulating starch solution. Immobilization of either amylase or antiamylase IgG makes dissolution of the antigen-antibody bond difficult.     

Stability of immobilized frog epidermis tyrosinase

Purified frog epidermis tyrosinase was immobilized on the following supports: CNBr-Sepharose 4B, Enzacryl AA, Enzacryl AH, Enzacryl Polythiolactone, and Enzacryl Polyacetal. The enzyme was active on all supports except when Enzacryl Polyacetal was used. The stability increased on immobilization. Enzacryl AA was the best support assayed. The Enzacryl AA-enzyme complex was 30- to 40-fold more stable to inactivation reaction than soluble enzyme, and maintained its activity when stored and assayed repeatedly. The immobilized enzyme on the other supports was also more stable than the soluble form. The pH-activity profile, thermal stability, storage stability, and the effect of protein concentration on activity of the immobilized enzyme have been studied. The properties observed for the immobilized enzyme were different than those of the soluble enzyme. The main reason for this difference could be due to enzyme modification through tyrosine groups of the enzyme; to conformational changes produced in the union to the matrix; and to microenvironmental differences created by the matrix.     

Synthesis of the bifuncttonal dinucleotide AMP-ATP and its application in general ligand affinity chromatography

A new AMP derivative substituted with spacer arms both at position N⁶ and C8 of the adenine moiety was synthesized and immobilized to Sepharose. To the immobilized ligand was subsequently coupled C8-substituted ATP in a solid-phase synthesis fashion yielding the bifunctional general ligand AMP-ATP. This affinity material was used in the separation of two major groups of enzymes, dehydrogenases and kinases. It was found that on passage of crude homogenates obtained from mouse kidney through the affinity column, several dehydrogenases and kinases were bound, which could be eluted separately using pulses of NADH and ATP, respectively. In the fractions obtained on NADH elution, lactate dehydrogenase, malate dehydrogenase, and α-glycerol phosphate dehydrogenase were found, whereas ATP eluted 3-phosphoglyceric acid kinase, pyruvate kinase, and aldolase.     

Facile formation of stimuli-responsive,fluorescent and magnetic nanoparticles based on cellulose stearoyl ester via nanoprecipitation

In comparison to stimuli-responsive, multi-functional nanoparticles (NPs) from synthetic polymers, such NPs based on sustainable, naturally occurring polysaccharides are still scarce. In the present study, stable stimuli-responsive, fluorescent and magnetic NPs were fabricated using cellulose stearoyl esters (CSEs) consisting of cellulose and stearoyl groups. The multifunctional NPs with the average diameters between 80 and 250 nm were obtained after facile nanoprecipitation using CSE solutions containing Fe₃O₄-NPs. Using the aqueous solution of fluorescent rhodamine B as precipitant, NPs with rhodamine B on NP surface were obtained. Rhodamine B could be released depending on the temperature. In comparison, stearoylaminoethyl rhodamine B can be encapsulated in CSE-NPs, which renders obtained NPs reversible fluorescence in response to UV illumination and heat treatment.     

Affinity chromatography ofd-lactate dehydrogenases fromLimulus polyphemus (horseshoe crab) andHaliotus sp. (abalone) voluntary muscle on 8-(6-aminohexyl)-amino-adenine nucleotide-sepharose

An improved method of purification employing sequential isoenzyme elution on DEAE-cellulose at pH 6.5 and biologically specific elution with the reduced NAD-pyruvate adduct from 8-(6-aminohexyl)-amino-NAD⊕-Sepharose is described forLimulus (horseshoe crab) muscle D- lactate dehydrogenase. The protein is judged as being at least 98%pure by its constant specific activity in the terminal purification steps, a molar extinction coefficient (280nm) identical with that previously reported for the purified enzyme, and its protein and enzyme electrophoretic patterns on starch and polyacrylamide gel electrophoresis at three pHs. The binding of ammonium sulfate fractionated D- lactate dehydrogenase from crude cell- free Limulus andHaliotus (abalone) muscle homogenates to 8-(6-aminohexyl)-amino-AMP-and 8-(6-aminohexyl)-amino-NAD®-Sepharose columns is demonstrated. A comparison of the binding properties of these two enzymes with those of vertebrate L-lactate dehydrogenases suggests that they may be significantly different in terms of their binding sites for the adenine portion of the coenzyme.     

Use of an affinity immunosorbent for the isolation of lactoferrin from breast milk serum

An affinity or biospecific method of isolating an iron-binding glycoprotein — lactoferrin — from breast milk is described which permits in a single chromatographic operation the isolation of lactoferrin homogeneous according to the results of electrophoresis in polyacrylamide gel. The affinity method of isolating lactoferrin is based on the use of a specific immunosorbent consisting of antibodies to lactoferrin covalently conjugated with cyanogen-bromide-activated Sepharose 4B.     

Immobilized citrate synthase

Kinetic properties of pig heart citrate synthase immobilized on Sepharose were determined. Compared to the free enzyme the K_m values for both acetyl-CoA and oxalacetate were increased. The kinetic pattern of the Lineweaver-Burk plots of both substrates for the immobilized enzyme was that of lines intersecting on thex axis. This is the same as that obtained for the free enzyme and indicates that there is no change in the kinetic mechanism of the reaction. The pH response and the Arrhenius plot of both the immobilized and free enzyme were the same. The enzymes show a break in their Arrhenius plots. The immobilized enzyme exhibits greater heat stability than does the free enzyme.     

Impact of PS/SiO2 morphologies on the SERS activity of PS/SiO2/Ag nanocomposite particles

To understand the relationship between the morphology of carboxyl-functionalized polystyrene/silica (PS/SiO₂) nanocomposite microspheres and the surface-enhanced Raman scattering (SERS) performance of PS/SiO₂/Ag nanocomposite particles, core-shell and raspberry-like PS/SiO₂ composite microspheres were used as templates to prepare PS/SiO₂/Ag nanocomposite particles. The core-shell and raspberry-like structured PS/SiO₂ templates were prepared via in situ sol-gel reaction by hydrolysis tetraethyl orthosilicate (TEOS) in alkali solution. Silver nanoparticles (10–50 nm) were loaded on the PS/SiO₂ templates’ surface by chemical reduction. The morphology and structure of the PS/SiO₂/Ag particles were characterized by TEM, SEM, X-ray diffraction (XRD), and ultraviolet-visible (UV-vis) spectroscopy. Rhodamine 6G (R6G) was selected as a model chemical to study the enhancement performance of substrate constructed by PS/SiO₂/Ag nanocomposite. Results indicated that the PS/SiO₂/Ag nanocomposite prepared based on the core-shell templates showed higher SERS activity. The beneficial effect was associated with a lower specific area of core-shell structure and the larger average diameter of nanosilvers than that of the raspberry-like templates.     

Heat of reaction and curing of epoxy resin

The heat of reaction and kinetics of curing of diglycidyl ether of bisphenol-A (DGEBA) type of epoxy resin with catalytic amounts of ethylmethylimidazole (EMI) have been studied by differential power-compensated calorimetry as a part of the program for the study of process monitoring for composite materials. The results were compared with those from 1∶1 and 1∶2 molar mixtures of DGEBA and EMI. A method of determination of heat of reaction from dynamic thermoanalytical instruments was given according to basic thermodynamic principles. The complicated mechanism, possibly involving initial ionic formation, has also been observed in other measurements, such as by time-domain dielectric spectroscopy. The behavior of commercially available DGEBA resin versus purified monomeric DGEBA were compared. The melting point of purified monomeric DGEBA crystals is 41.4 °C with a heat of fusion of 81 J/g. The melt of DGEBA is difficult to crystallize upon cooling. The glass transition of purified DGEBA monomer occurs around ?22 °C with aΔC _p of 0.60 J/K/g.     

Flavin-protein interaction in bound glucose oxidase

Glucose oxidase was bound to Sepharose, Sephadex, gelatin, and dextran, yielding immobilized soluble and insoluble derivatives of the enzyme. The soluble preparations possessed higher enzymic activity than the analogous insoluble ones. The reversible dissociation process of the bound enzyme into apoenzyme and flavin adenine dinucleotide (FAD) was studied with the soluble and insoluble glucose oxidase in relation to enzymic activity and conformational changes as measured by circular dichroism and fluorescence methods. Bound apoenzyme was found to be more stable than the apoenzyme obtained from the unmodified glucose oxidase. The binding constant of FAD in bound glucose oxidase (K_diss≈10^-8M) calculated from fluorescent studies was lower than that of FAD in the native enzyme (K_diss10^-10M). The circular dichroism measurements indicated that dextran-bound glucose oxidase has a conformation similar to that of the native enzyme.     

Reduction in the antigen-binding in polyacrylamide gel

Under conditions of antibody excess, antibodies that had been immobilized by physical entrapment in a polyacrylamide gel matrix consistently removed less radiolabeled microprotein antigen from solution than did the same antibodies chemically conjugated to Sepharose beads. This reaction is believed to relate to the difficulty of formation of secondary antibody-antigen lattices when the molecules are held in “cells” in the polymer matrix.