Inhibition of horseradish peroxidase byN-ethylamide ofo-sulfobenzoylacetic acid

The carboxylic groups of horseradish peroxidase were modified by 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide metho-p-toluenesulfonate by the Koshland method. The catalytic properties of the native and modified peroxidase were studied in the presence ofN-ethylamide ofo-sulfobenzoylacetic acid (EASBA) at pH 5.0–7.5. In the oxidation ofo-dianisidine, EASBA is a competitive inhibitor of the carbidiimide-modified peroxidase, and it increases bothK _m andV _m in the case of the native enzyme. These data show that at least one of the carboxylic groups modified with carbodiimide is located at the area of the peroxidase active site.     

Effect of solid-phase chemical modification on the features of the lipase from Thermomyces lanuginosus

The lipase from Thermomyces lanuginosus (TLL) was immobilized on octyl Sepharose and further modified with ethylenediamine (EDA) after activation of the carboxylic groups with carbodiimide. Different degrees of modification of the carboxyl groups were carried out by controlling the concentration of carbodiimide (10%, 50% or 100%). Subsequently, the effect of incubation of the modified preparations on hydroxylamine to recover the modified tyrosine was also studied. The modified enzymes exhibited a mobility in native electrophoresis quite different from that of the unmodified lipase (as expected by the changes in charge), and required higher concentrations of cationic detergent to become desorbed from the support. Interestingly, the chemical modification of the immobilized TLL produced an improvement in its activity, proportional to the amination degree. This increase in activity was much more significant at pH 10, where the fully modified preparation increased the activity by a factor of 10 as compared to the unmodified preparation. Moreover, the incubation of the chemically aminated preparations in a hydroxylamine solution improved the activity by an additional factor of 1.2. The fully aminated and incubated in hydroxylamine preparation exhibited a thermostability higher than that of the unmodified preparation, mainly at pH 5 (almost a 30 fold factor). In the presence of tetrahydrofurane, some stabilization was observed at pH 7, while at pH 9 the stability of the modified enzyme decreased (under all the assayed amination degrees) when compared to that of the unmodified enzyme. Thus, this simple protocol may be a rapid and efficient way of preparing a TLL biocatalyst with higher activity and stability, although this will depend on the inactivation conditions.     

The reaction of hydrogen peroxide with the dimethyl ester heme derivative of cytochrome c peroxidase

A modified cytochrome c peroxidase was prepared by reconstituting apocytochrome c peroxidase with protoheme in which both heme propionic acid groups were converted to the methyl ester derivatives. The modified enzyme reacted with hydrogen peroxide with a rate constant of (1.3 +/- 0.2) x 10(7) M-1 s-1, which is 28% that of the native enzyme. The reaction between the modified enzyme and hydrogen peroxide was pH-dependent with an apparent pK of 5.1 +/- 0.1 compared to a value of 5.4 +/- 0.1 for the native enzyme. These observations support the conclusion that the apparent ionization near pH 5.4, which influences the hydrogen peroxide-cytochrome c peroxidase reaction is not due to the ionization of the propionate side chains of the heme group in the native enzyme. A second apparent ionization, with pK of 6.1 +/- 0.1, influences the spectrum of the modified enzyme which changes from a high spin type at low pH to a low spin type at high pH.     

Peroxidase activity of hemoglobin, modified at the carboxylic groups of heme and amino acids]

The effects of modification of heme carboxylic groups by omega-aminoenantic acid and L-phenylalamine on the peroxidase activity of hemoglobin were studied. For this purpose the peroxidase activities of the original compounds--hemin, hemin-aminoenantic acid, hemin-phenylalanine and hemoglobins prepared from the hemin and globin compounds--hemoglobin, aminoenantyl-hemoglobin and phenylalanine hemoglobin--were determined. The dependence of the peroxidase activity of these compounds on their concentrations and pH was analyzed. It was shown that 40--50% modification of the heme carboxylic groups by amino acids decreases the peroxidase activity of the modified hemins and that of modified hemoglobins reconstructed from these hemins and globin. A decrease of the catalytic activity of the hemoglobin derivatives is due to a lower peroxidase activity (as compared to hemin) of the modified hemins. It is thus concluded that the amino acid modification of the carboxylic groups of heme does not affect the heme-protein interactions in the hemoglobin molecule.     

Polymeric grafting of acrylic acid onto poly(3-hydroxybutyrate-co-3-hydroxyvalerate): surface functionalization for tissue engineering applications

Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) melt processed disks and solvent cast films were modified by graft co-polymerization with acrylic acid (AAc) in methanol solution at ambient temperature using gamma irradiation (dose rate of 4.5 kGy/h). To assess the presence of carboxylic acid groups on the surface, reaction with pentafluorophenol was performed prior to X-ray photoelectron spectroscopy analysis. The grafting yield for all samples increased with monomer concentration (2-15%), and for the solvent cast films, it also increased with dose (2-9 kGy). However, the grafting yield of the melt processed disks was largely independent of the radiation dose (2-8 kGy). Toluidine blue was used to stain the modified materials facilitating visual information about the extent of carboxylic acid functionalization and depth penetration of the grafted copolymer. Covalent linking of glucosamine to the functionalized surface was achieved using carbodiimide chemistry verifying that the modified substrates are suitable for biomolecule attachment.     

Neocarzinostatin: effect of modification of side chain amino and carboxyl groups on chemical and biological properties.

The antitumor protein neocarzinostatin (NCS), isolated from Streptomyces carzinostaticus, is a single chain polypeptide with 109 amino acid residues. Complete acylation of the amino groups (alanine-1 and lysine-20) was observed when NCS was allowed to react with 3-(4-hydroxyphenyl)-propionic acid N-hydroxysuccinimide ester at pH 8.5. Since the ensuing bis[(alanine-1, lysine-20)-3-(4-hydroxyphenyl)]-propionamide NCS was fully active in antibacterial potency and in the inhibition of growth of leukemic (CCRF-CEM) cells in vitro, it appears that the two amino groups in the protein are not essential for biological activity. Radiolabeled NCS was prepared by using a tritiated or 125I-labeled acylating agent. Since the CD spectra of native and bis(alanine-1, lysine-20)-amino modified NCS were indistinguishable, there is presumably no change in the native conformation of the protein due to acylation. Reaction of NCS with ammonium chloride in the presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide at pH 4.75 converted all the 10 carboxyl groups into carboxamides and produced a protein derivative of basic character. This modification caused a change in the native conformation of the protein accompanied by a loss in biological inhibitory activities.     

Chemical modification of lactase for immobilization on carboxylic acid-functionalized microspheres

Abstract

Surface interactions between an enzyme and support influence the retention of activity after immobilization. Chemical modification of enzymes prior to immobilization may be used to alter these interactions and enhance activity retention. Lactase (A. oryzae) was covalently conjugated to P(S/V-COOH) microspheres, with surface carboxylic acid densities of 9 μeq/g and 137 μeq/g, using carbodiimide chemistry. Under optimum pH and temperature conditions, activity retention was greater when the enzyme was conjugated to microspheres containing a lower density of surface carboxylic acid groups (32% activity retention) than when the enzyme was conjugated to microspheres having a greater density of surface carboxylic acid groups (11% activity retention). Chemical modification of lactase carboxylic acid groups with glucosamine prior to immobilization was evaluated as a means to increase activity retention. Under optimal conditions, modification resulted in a 17% decrease in soluble enzyme activity compared to the native enzyme. However, immobilization of the modified enzyme yielded 85% and 64% activity retention after conjugation to microspheres with a lower and higher density of surface carboxylic acid groups, respectively. The results suggest that increases in surface carboxylic acid density on the carrier promote the loss of lactase activity after immobilization, and chemical modification of the enzyme with glucosamine provides a means to retain catalytic activity after attachment to these supports.     

Formation of a novel heparin-based hydrogel in the presence of heparin-binding biomolecules

An injectable, heparin-based hydrogel system with the potential to be gelled with cells was developed. First, heparin was modified to have thiol groups by the modification of carboxylic groups of heparin with cysteamine using carbodiimide chemistry. Thiol functionalization of heparin carboxylic groups was controlled from 10% to 60% of the available COOH groups, and the retained bioactivity of the modified heparin, characterized by its binding affinity to antithrombin III, decreased with increasing functionalization. Then, the thiol-functionalized heparin was reacted with poly(ethylene glycol) diacrylate to form a hydrogel. The gelation kinetics and mechanical properties of the final gel state could be tuned by controlling cross-link density. Fibroblast cell encapsulation using this hydrogel revealed the nontoxicity of the present system. Cell proliferation inside the hydrogel was observed, and it was significantly enhanced (more than 5-fold) by the addition of fibrinogen into the hydrogel during gelation.     

Increased thermostability and phenol removal efficiency by chemical modified horseradish peroxidase

Horseradish peroxidase was modified by phthalic anhydride and glucosamine hydrochloride. The thermostabilities and removal efficiencies of phenolics by native and modified HRP were assayed. The chemical modification of horseradish peroxidase increased their thermostability (about 10- and 9-fold, respectively) and in turn also increased the removal efficiency of phenolics. The quantitative relationships between removal efficiency of phenol and reaction conditions were also investigated using modified enzyme. The optimum pH for phenol removal is 9.0 for both native and modified forms of the enzyme. Both modified enzyme could suffer from higher temperature than native enzyme in phenol removal reaction. The optimum molar ratio of hydrogen peroxide to phenol was 2.0. The phthalic anhydride modified enzyme required lower dose of enzyme than native horseradish peroxidase to obtain the same removal efficiency. Both modified horseradish peroxidase show greater affinity and specificity of phenol.     

Chemical modification of lysine epsilon-NH2-groups in horseradish peroxidase. Its effect on enzyme stability. Temperature dependence of thermo-inactivation constants for native and modified peroxidase]

Thermostability of horseradish peroxidase modified by acetic, propionic, butyric, valeric and succinic anhydrides and trinitrobenzolsulfonic acid (TNBS) is studied within the temperature range of 56-80 degrees C. Acylation of 4 amino groups and arylation of 3 amino groups with TNBS are found to stabilize the enzyme, while modification of 6 groups decreases the enzyme stability. Chemical modification of peroxidase does not change its pH-dependence with respect to enzyme thermostability. Thermodynamic activation parameters of irreversible thermoinactivation are determined for native and modified peroxidase. Native peroxidase has deltaH not equal to = 30+/-1 kcal/mole and deltaS not equal to = 14 e. e.; modified by acid anhydrides peroxidase has deltaH not equal to within 64-87 kcal/mole and deltaS not equal to within 110-178 e. e. depending on the nature of a modifying agent. The effect of the structure of a radical introduced into the enzyme molecule, and of a number of modified epsilon-amino groups on thermoinactivation deltaH not equal to and deltaS not equal to values is discussed.     

Chemical modification of epsilon-amino lysine groups in horseradish peroxidase. Its effect on catalytic properties and spatial structure of the enzyme]

Effect of chemical modification of horseradish peroxidase lysine epsilon-amino groups by propionic, butyric, valeric, succinic anhydrides and trinitrobenzolsulfonic acid (TNBS) on catalytic properties of the enzyme is investigated. All the preparations of modified peroxidase have 100% peroxidase activity for o-dianizidine at pH 7.0, which indicates the absence of lysine epsilon-amino group in the enzyme active site. pH-dependencies of modified peroxidase relative activity are studied; modification by anhydrides of monobasic acids is not found to result in changes of the relative activity pH-profile, while modification by succinic anhydride widens it. Absorption and circular dichoism spectra of native and modified peroxidase within 260--270 nm are obtained, some changes in the enzyme tertiary structure after its epsilon-amino groups modification are observed. Modification of four epsilon-amino groups by buturic and succinic anhydrides and of three epsilon-amino groups by TNBS is found to increase the regidity of protein surrounding of heme, and modification of six epsilon-amino groups by TNBS results in more unwrapped enzyme structure as compared with its native molecule.     

Identification of specific carboxyl groups on Anabaena PCC 7119 flavodoxin which are involved in the interaction with ferredoxin-NADP+ reductase.

Flavodoxin from the nitrogen-fixing cyanobacteria Anabaena PCC 7119 forms an electron-transfer complex with ferredoxin--NADP+ reductase (FNR) from the same organism. The complex is mainly governed by electrostatic interactions between side-chain amino groups of the reductase and carboxyl residues of flavodoxin. In order to localize the binding site on flavodoxin, chemical modification of its carboxyl groups has been carried out. Treatment of flavodoxin with a water-soluble carbodiimide, N-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), in the presence of a nucleophile, glycine ethyl ester, caused a time-dependent modification of the protein that is responsible for the loss of its ability to participate as electron carrier in the photoreduction of NADP+ by chloroplast membranes, and also in NADPH--cytochrome-c reductase activity, by about 85%. Nevertheless, the ability of flavodoxin to receive electrons from the reducing side of photosystem I was much less affected. The inhibition was enhanced at low pH, suggesting that carboxylic acid groups were the target of chemical modification. Treated flavodoxin failed to form covalent complexes with FNR and the dissociation constant for the non-covalent complex with FNR was fourfold higher. After tryptic digestion of a sample of flavodoxin modified by EDC in the presence of [1-14C]glycine ethyl ester, two major radioactive peptides were isolated. The first protein fragment contained three carboxylic residues (Asp123, Asp126 and Asp129), corresponding to the region where long-chain flavodoxins show an insert compared to short-chain flavodoxins. The second peptide corresponded to a similar region, either in the amino acid sequence or in the three-dimensional structure of the protein and also containing three carboxyl groups (Asp144, Glu145 and Asp146). Four of these carboxyl groups (Asp123, Asp126, Asp144 and Asp146) are highly conserved in all long-chain flavodoxins, suggesting that they could play an essential role in substrate recognition.     

Improved stabilization of chemically aminated enzymes via multipoint covalent attachment on glyoxyl supports

The surface carboxylic groups of penicillin G acylase and glutaryl acylase were chemically aminated in a controlled way by reaction with ethylenediamine via the 1-ethyl-3-(dimethylamino-propyl) carbodiimide coupling method. Then, both proteins were immobilized on glyoxyl agarose. In both cases, the immobilization of the chemically modified enzymes improved the enzyme stability compared to the stability of the immobilized but non-modified enzyme (by a four-fold factor in the case of PGA and a 20-fold factor in the case of GA). The chemical modification presented a deleterious effect on soluble enzyme stability. Therefore, the improved stability should be related to a higher multipoint covalent attachment, involving both the lysine amino groups and also the new amino groups chemically introduced on the enzyme. Moreover, the lower pK(a) of the new amino groups permitted to immobilize the enzyme under milder conditions. In fact, the aminated proteins could be immobilized even at pH 9, while the non-modified enzymes could only be immobilized at pH over 10.     

Chemical modification results in hyperactivation and thermostabilization of Fusarium solani glucoamylase

Chemical modification of carboxyl groups of glucoamylase from a mesophilic fungus, Fusarium solani, was carried out using ethylenediamine as nucleophile in the presence of water-soluble 1-ethyl-3(3-dimethylaminopropyl)carbodiimide. Modification brought about a dramatic enhancement of catalytic activity and thermal stability of glucoamylase. Temperature and pH optima of ethylenediamine-coupled glucoamylase (ECG) increased as compared with those of native enzyme. The specificity constant (k(cat)/K(m)) of native, ECG-2, ECG-11, and ECG-17 was 136, 173, 225, and 170, respectively, at 55 degrees C. The enthalpy of activation (Delta H*) and free energy of activation (Delta G*) for soluble starch hydrolysis were lower for the chemically modified forms. All of the modified forms were stable at higher temperatures and possessed high Delta G* against thermal unfolding. The effects of alpha-chymotrypsin and subtilisin on the modified forms were activating as compared with native. Moreover, denaturation of ECG-2, ECG-11, and ECG-17 in urea at 4 mol x L(-1) also showed an activation trend. A possible explanation for the thermal denaturation of native and increased thermal stability of ECG-2, ECG-11, and ECG-17 at higher temperatures is also discussed.     

Optimization of reaction conditions during enzyme immobilization on soluble carboxyl-containing carriers

The kinetics of 1-ethyl-3-(3′-dimethylaminopropyl)-carbodiimide interaction with carboxylic groups in low and high molecular weight compounds have been investigated for choosing the optimum conditions for enzyme immobilization on carboxylic carriers. Optimum pH values depended on the pK_a of an acid. The ionic strength of the reaction mixture influenced the process of activation with carbodiimide and a decrease in reaction rate accompanied an increase in ionic strength. Optimum conditions for the coupling process with the use of carbodiimide activation are suggested. The process of fibrinolysin immobilization on the soluble copolymer of acrylic acid and acrylamide with carbodiimide as a coupling agent was also studied.     

Conformational change of turkey-gizzard caldesmon induced by specific chemical modification with carbodiimide

Water soluble 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide was used to internally cross-link carboxyl and lysyl groups of caldesmon. The modification did not involve the two cysteines of the molecule which were previously labelled with N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine. The modified caldesmon exhibited a smaller Stokes radius (4.0 nm instead of 6.3 nm) and its electrophoretic mobility corresponded to an apparent molecular mass of approximately 82 kDa, appreciably lower than that of the native molecule (120 kDa), but more similar to the reported true molecular mass of 86,974 Da of chicken-gizzard caldesmon (Bryan, J., Imai, M., Lee, R., Moore, P., Cook. R. G. & Lin, W. (1989) J. Biol. Chem. 264, 13,873-13,879). Comparative circular dichroism analysis indicated a decrease of the alpha-helix content from 43% to 36% resulting from the chemical modification. The 1H-NMR spectra of the native and modified caldesmon showed that the covalent cross-linking affected mainly the central and N-terminal parts of the molecule. The C-terminal part, rich in aromatic amino acids, was unmodified by the carbodiimide treatment. This was also corroborated by the continued ability of the modified caldesmon to bind to actin and calmodulin, and by the property of the 90-kDa proteolytic N-terminal fragment to give an internally cross-linked species of 60 kDa. Using electron microscopy, the modified protein was shown to have a more compact shape and a reduced capacity to induce tight and long F-actin bundles. These conformational changes were obtained when the carbodiimide reaction was conducted at pH 6.0 and were not observed at pH 8.0. This suggests that local variation of the pH might affect the conformation of caldesmon which changes from an elongated to more compact shape, stabilized by electrostatic interactions. It is proposed that the flexibility of caldesmon might be involved in the regulatory function of this protein in the smooth muscle and might favour tightly packed F-actin bundles or weaker interactions between actin filaments.     

Chemical modification of carboxyl groups of fibrinogen and its effect on the binding of cationic detergent.

Carboxyl groups of native human fibrinogen were modified with glycine methyl ester and 1-ethyl-3(3-dimethylaminopropyl)carbodiimide. It seemed likely that the modification occurred stepwise. Approximately 26% of the carboxyl groups of fibrinogen was modified finally. The modified fibrinogen had no interaction with cationic detergent, and did not form any complex with the detergent. In dilute acid, fibrinogen was observed to show only a slight interaction with cationic detergent. It is probable that the exposed and ionized carboxyl groups are essential for the formation of a complex between fibrinogen and cationic detergent.     

Synthesis of new polymer-bound adenine nucleotides using starburst PAMAM dendrimers

Two types of new polymer-bound adenine nucleotides were synthesized by coupling adenine nucleotides (ATP and ADP) with starburst polyamidoamine (PAMAM) dendrimers. The first type was obtained by coupling native adenine nucleotides directly with a carboxy-terminated PAMAM dendrimer. In the second type, the nucleotides were modified by introducing a spacer arm containing a carboxylic end group (N(6)-R-ATP and N(6)-R-ADP) and coupled with an amine-terminated PAMAM dendrimer. Both types of the dendrimers were coupled with native or the modified nucleotides using the well-known carbodiimide activation technique. The optimum coupling pH and temperature were 4 and 30 degrees C, respectively, for preparing the carboxy-terminated PAMAM-bound ATP or ADP, and were 9 and 50 degrees C, respectively, for preparing the amine-terminated PAMAM-bound N(6)-R-ATP or N(6)-R-ADP. The ATP or ADP contents in the synthesized polymers were found to be 4 mol of ATP or of ADP/mol of carboxy-terminated PAMAM-bound ATP or ADP and 25 mol of ATP or of ADP/mol of amine-terminated PAMAM-bound N(6)-R-ATP or N(6)-R-ADP. The coenzymatic activities relative to the native ATP of the carboxy-terminated PAMAM-bound ATP against glucokinase and hexokinase were 16 and 7%, respectively, and those of the amine-terminated PAMAM-bound N(6)-R-ATP 2 and 1%, respectively. The coenzymatic activities relative to the native ADP of the carboxy-terminated PAMAM-bound ADP and the amine-terminated PAMAM-bound N(6)-R-ADP against acetate kinase were 24 and 3.5%, respectively.     

Thrombolytic action of urokinase preparation covalently bound to modified thrombin

Urokinase was covalently bounded with modified thrombin. Thrombin was modified by carbodiimide and 1, 12-dodecamethylenediamine. In this conjugate thrombin is not catalytically active and does not induce platelets aggregation. The catalytic properties of modified urokinase do not essentially differ from native enzyme but its thermostability increases. The modified urokinase thrombolytic effect is at least 10-fold higher than the native one. In femoral arteries of experimental thrombosis the conjugate urokinase-thrombin brings about total thrombolytic effect as early as 1.5 hours after injection (2500 IU per 1 kg of the animals weight). The causes of the observed effect were discussed.     

Controlled layer-by-layer immobilization of horseradish peroxidase.

Horseradish peroxidase (HRP) was biotinylated with biotinamidocaproate N-hydroxysuccinimide ester (BcapNHS) in a controlled manner to obtain biotinylated horseradish peroxidase (Bcap-HRP) with two biotin moieties per enzyme molecule. Avidin-mediated immobilization of HRP was achieved by first coupling avidin on carboxy-derivatized polystyrene beads using a carbodiimide, followed by the attachment of the disubstituted biotinylated horseradish peroxidase from one of the two biotin moieties through the avidin-biotin interaction (controlled immobilization). Another layer of avidin can be attached to the second biotin on Bcap-HRP, which can serve as a protein linker with additional Bcap-HRP, leading to a layer-by-layer protein assembly of the enzyme. Horseradish peroxidase was also immobilized directly on carboxy-derivatized polystyrene beads by carbodiimide chemistry (conventional method). The reaction kinetics of the native horseradish peroxidase, immobilized horseradish peroxidase (conventional method), controlled immobilized biotinylated horseradish peroxidase on avidin-coated beads, and biotinylated horseradish peroxidase crosslinked to avidin-coated polystyrene beads were all compared. It was observed that in solution the biotinylated horseradish peroxidase retained 81% of the unconjugated enzyme's activity. Also, in solution, horseradish peroxidase and Bcap-HRP were inhibited by high concentrations of the substrate hydrogen peroxide. The controlled immobilized horseradish peroxidase could tolerate much higher concentrations of hydrogen peroxide and, thus, it demonstrates reduced substrate inhibition. Because of this, the activity of controlled immobilized horseradish peroxidase was higher than the activity of Bcap-HRP in solution. It is shown that a layer-by-layer assembly of the immobilized enzyme yields HRP of higher activity per unit surface area of the immobilization support compared to conventionally immobilized enzyme.