Analysis and optimization of methods using water-soluble carbodiimide for immobilization of biochemicals to porous glass

Two methods employing a water-soluble carbodiimide for carboxyl activation were investigated for the immobilization of biochemicals to succinamidopropyl-porous glass beads. Immobilization using the simultaneous method (simultaneous addition of carbodiimide and nucleophilic ligand to the beads) and large excess of carbodiimide and a small nucleophile should result in covalent binding to all accessible carboxyl groups. Results obtained with glycine methyl ester indicated that 40% of the total surface carboxyl groups were sterically accessible. Using these reaction conditions with the protein, chymotrypsinogen, suggests that a surface monolayer is immobilized. although far fewer sites are required assuming single point attachment. For ligands containing carboxyl groups and several nucleophilic groups (e. g., enzymes), however, biological inactivation may occur using the simultaneous method. Consequently, a sequential method (activation of the surface with carbodiimide followed by washing and addition of the biochemical to be immobilized) was optimized. Using optimal conditions (20 min activation time at pH 4.75 and room temperature; 2 min wash at pH 7 and 0 degrees C) and 0.1M carbodiimide, nearly half of the accessible surface sites remained in the O-acylisourea form and reacted with glycine methyl ester upon its addition. The amount of surface loading as a function of activation time was consistent with a model constructed using rate constants for O-acylisourea formation and hydrolysis previously derived from solution studies with acetic acid [Swaisgood and Natake, J. Biochem 74, 77 (1973)]. Measurement of reaction rates with glycine methyl ester following surface activation suggests that the rate of reaction with amino groups is at least eightfold greater than the hydrolysis rate. Either immobilization procedure gave comparable enzyme loading and specific activities for the case of sulfhydryl oxidase.     

Carboxyl groups at the two active centers of sucrase-isomaltase from rabbit small intestine.

1. Seveal selective reagents were employed to identify the amino acid residues essential for the catalytic activity of sucrase-isomaltase. 2. Modification of histidine, lysine and carboxyl residues resulted in a partial inactivation of the enzyme. Substrates or competitive inhibitors provided protection against inactivation only in the reaction of carboxyl groups with carbodiimide (+lycine ethyl ester) or with diazoacetic ethyl ester. This indicated the occurrence of carboxyl groups at the two active centers of the enzyme complex. 3. Protection against inactivation of the enzyme by carbodiimide was provided also by the presence of alkali and alkaline earth metal ions, which are non-essential activators of sucrase-isomaltase. The presence of Na+ and Ba2+ protected approximately one carboxyl group per active center from reacting with carbodiimide plus glycine ethyl ester. 4. The carbodiimide-reactive groups were not identical with the two carboxylate groups recently found to react with conduritol-B-epoxide, an active-site-directed inhibitor of sucrase-isomaltase (Quaroni, A. and Semenza, G., 1976, J. Biol. Chem 251,3250--3253). A possible role for the carbodiimide-reactive carboxyl groups at the active centers of sucrase-isomaltase is discussed.     

A radiochemical approach to the determination of carboxylic acid groups in polysaccharides

A method for the routine radiochemical determination of carboxylic acid groups in polysaccharides is described. The procedure is based on sodium borotritide reduction of the product of the reaction between water-soluble carbodiimide derivatives and carboxyl groups on the polysaccharide. Carboxymethyl-cellulose and hyaluronic acid were used as test polysaccharides.     

Introduction of sulfhydryl groups into proteins at carboxyl sites

A two-step procedure for introduction of sulfhydryl groups at protein carboxyl groups is described. The resultant proteins contain 2-aminoethanethiol residues bound by amide linkages to the protein carboxyl groups. First an amide bond is formed between a carboxyl group of the protein and one of the amino groups of cystamine. Then the disulfide bond is reduced with dithiothreitol, yielding the amide of 2-aminoethanethiol. This procedure was used to incorporate sulfhydryl groups into carbonic anhydrase and adrenocorticotropic hormone. The effect of carbodiimide concentration and pH of the coupling reaction on stoichiometry of sulfhydryl group incorporation was examined. The method was used to prepare bovine carbonic anhydrase containing up to nine sulfhydryl groups per molecule with no loss of enzymatic activity and biologically active adrenocorticotropic hormone containing one sulfhydryl group per molecule.     

Use of regularly structured bacterial cell envelope layers as matrix for the immobilization of macromolecules

Summary Carboxyl groups present on the outer face of the hexagonally ordered S-layer lattices from Bacillus stearothermophilus PV72 and Clostridium thermohydrosulfuricum L111-69 were activated with carbodiimide. The reaction of the activated carboxyl groups with free amino groups of low molecular weight nucleophiles was controlled by labelling with polycationized ferritin, a net positively charged topographical marker for electron microscopy, which densely binds to S-layers possessing free carboxyl groups. Carbodiimide-activated carboxyl groups were also allowed to react with amino groups of ferritin (MW 440 000) and invertase (MW 270 000). Covalent attachment of ferritin was examined by electron microscopy. Using invertase, approximately 1 mg enzyme was bound per mg S-layer protein indicating a high packing density of invertase molecules on the outer face of the S-layer lattice. The immobilized invertase retained 70% of its original activity.     

Use of Water-soluble Carbodiimide (EDC) for Immobilization of EDC-sensitive Dextranase

Water-soluble carbodiimide [EDC: (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide)] is a useful reagent for chemical modification of carboxyl group of various proteins. Model experiments to establish detailed conditions for the cross-linking reaction with EDC were conducted. Since the reactivity of hexamethylenediamine as a nucleophile was almost comparable to that of glycine ethyl ester, AH-Sephadex and the carboxyl group of aspartylphenylalanine methyl ester were coupled by EDC. From the hydrolyzate of the isolated gel, aspartic acid and phenylalanine methyl ester were identified. When bovine serum albumin (BSA) was incubated with AH-Sephadex and EDC, about 90 % of the BSA was coupled to the gel by 3 hr incubation. Moreover, BSA was effectively coupled with the carboxymethyl cellulose (CMC) after activation of the carboxyl groups of CMC with EDC followed by the removal of excess EDC. The latter case would be useful for cross-linking the enzyme molecules to the matrix because of the very mild reaction conditions. For example, endodextranase, which readily lost its activity upon being incubated with EDC (suggesting that a carboxyl group was essential for the enzyme activity), was effectively immobilized to CMC with EDC. This improved reaction step for the cross-linking seemed to be especially useful for the glycosylases, because in most of these enzymes carboxyl groups play a role in the catalytic residue.     

Investigations of dye binding in the sequential staining of mucosaccharides by Alcian Yellow-Alcian Blue. 1. Histochemical experiments on different animal and human mucosaccharides

Synopsis It may be assumed that, histochemically, carboxyl groups and sulphate half-ester groups of muco electrolyte concentration of the dye baths in the two steps of a sequential Alcian Yellow-Alcian Blue method. In the present study the specificity and reliability of this method has been investigated.When the staining conditions were the same in both steps, the second dye (Alcian Blue) was found to stain mucosubstances in spite of the prior staining with Alcian Yellow. Binding of Alcian Blue was observed in all but very dilute Alcian Blue solutions. The degree of Alcian Blue binding depended on the dye concentration and staining time of the second step (Alcian Blue), and it varied widely for different mucosubstances. Although an incomplete saturation of anionic groups with dye molecules in the first step cannot be completely excluded, it is thought that Alcian Blue displaces Alcian Yellow from the carboxyl and sulphate groups of mucosubstances in tissue sections.It seems that the sequential Alcian Yellow-Alcian Blue method, under the conditions investigated, does not provide a reliable means for differentiating carboxyl and sulphate groups of mucosbstances in tissue sections simultaneously, because the second dye obviously is capable of displacing the first dye from sulphate groups. However, it is possible to distinguish non-sulphated acid mucosaccharides from sulphated mucosaccharides.     

The essential activated carboxyl group of inorganic pyrophosphatase.

1. A carboxyl group of high reactivity has been found in inorganic pyrophosphatase (pyrophosphate phosphohydrolase, EC 3.6.1.1) from yeast. This group interacts with agents which react neither with carboxyl groups of low molecular weight compounds nor with other carboxyl groups of the protein. 2. The reaction of this activated carboxyl group with inorganic phosphate, hydroxylamine, N-methyl- and O-methylhydroxylamines, and glycine methyl ester has been studied. 3. Homoserine and homoserine lactone were found in the hydrolyzate of phosphorylated and NaBH4-reduced pyrophosphatase, indicating that an aspartyl residue is phosphorylated. 4. Hydroxylamine and other nucleophilic agents cause inactivation of pyrophosphatase as a result of interaction with a carboxyl group. Both diaminobutyric and diaminopropionic acids were seen in the acid hydrolyzate of the protein treated with hydroxylamine and subjected to rearrangement in the presence of carbodiimide. 5. The ways in which the activation of a carboxyl group in the enzyme is achieved and the presumed mechanism of action of inorganic pyrophosphatase are discussed.     

Specific modification of carboxyl-groups in bovine growth hormone by a soluble carbodiimide and glycinemethylester

The reactivity of the carboxyl groups in bovine growth hormone was studied by reaction with 1-ethyl-3(3-dimethylaminopropyl) carbodiimide in the presence of an excess of glycinemethylester. Localization in the molecule of the various kinetically distinguishable carboxyl groups was achieved. Highest reactivity was found in carboxyl groups 30, 125, 127 and 128. These residues were followed, as to reactivity, by a set including carboxyl groups 65, 105, 151, 152, 185 and 190. Modification of approximately one fifth of the carboxyl groups in bovine growth hormone led to an important decrease in its growth promoting activity and capacity to compete with 125I-labeled growth hormone for rat liver binding sites. Demethoxylation restored most of the original biological activity.     

Chemical modification of a beta-glucosidase from Schizophyllum commune: evidence for essential carboxyl groups

The beta-glucosidase from Schizophyllum commune was purified to homogeneity by a modified procedure that employed Con A-Sepharose. The participation of carboxyl groups in the mechanism of action of the enzyme was delineated through kinetic and chemical modification studies. The rates of beta-glucosidase-catalyzed hydrolysis of p-nitrophenyl-beta-D-glucoside were determined at 27 degrees C and 70 mM ionic strength over the pH range 3.0-8.0. The pH profile gave apparent pK values of 3.3 and 6.9 for the enzyme-substrate complex and 3.3 and 6.6 for the free enzyme. The enzyme is inactivated by Woodward's K reagent and various water-soluble carbodiimides; chemical reagents selective for carboxyl groups. Of these reagents, 1-ethyl-3-(4-azonia-4,4-dimethylpentyl)carbodiimide iodide in the absence of added nucleophile was the most effective and a kinetic analysis of the modification indicated that one molecule of carbodiimide is required to bind to the beta-glucosidase for inactivation. Employing a tritiated derivative of the carbodiimide, 44 carboxyl groups in the enzyme were found to be labelled while the competitive inhibitor deoxynojirimycin protected three residues from modification. Treatment of the enzyme with tetranitromethane resulted in the modification of five tyrosine residues with approx. 28% diminution of enzymic activity. Titration of denatured enzyme with dithiobis(2-nitro-benzoic acid) indicated the absence of free thiol groups. Reaction of the enzyme with diethyl pyrocarbonate resulted in the modification of four histidine residues with the retention of 78% of the original enzymatic activity. The divalent transition metals Cu2+ and Hg2+ were found to be potent inhibitors of the enzyme, binding in an apparent irreversible manner.     

Native Enzyme Mobility Shift Assay (NEMSA): a new method for monitoring carboxyl group modification of carboxymethylcellulase from Aspergillus niger

A simple, sensitive, accurate and more informative assay for determining the number of modified groups during the course of carboxyl group modification is described. Monomeric carboxymethylcellulase (CMCase) from Aspergillus niger was modified by 1-ethyl-3(3-dimethylaminopropyl)carbodiimide (EDC) in the presence of glycinamide. The different time-course aliquots were subjected to non-denaturing PAGE and the gel stained for CMCase activity. The number of carboxyl groups modified are directly read from the ladder of the enzyme bands developed at given time. This method showed that after 75 min of modification reaction there were five major species of modified CMCases in which 6 to 10 carboxyls were modified.     

Some investigations of the mechanism of the so-called “methylation” reactions used in mucosubstance histochemistry

Summary The results reported in this paper substantiate Vilter's (1968) hypothesis that the absence of basophilia of acid mucosubstances after a so-called histochemical methylation with acidified methanol may be due to, among other factors, the lactonisation of carboxyl groups rather than, as is generally held, their esterification. Sections from several fixed tissues of Syrian hamster and mouse were treated at 60 ° or at room temperature with dry methanol, ethanol, butan-l-ol, benzene or cyclohexane containing 0.35% hydrogen chloride or 1% thionyl chloride. Others were treated with methanolic methyl iodide or etheral diazomethane. With most of these solutions, the azurophilia attributable to mucosubstance carboxyl and sulphate half-ester groups was eventually abolished; subsequent saponification restored only the azurophilia due to carboxyl groups. However, some variation was found for each methylating agent, and also between different mucosubstances.     

A single-step purification of rat pancreatic and salivary amylase by affinity chromatography

Optimal conditions for the conjugation of carboxyl groups on low molecular weight molecules to reactive amino groups on rabbit immunoglobulin G (IgG) using a modified carbodiimide reaction have been investigated. Reaction of [¹⁴C]hippuric acid with N-ethyl-N′-(dimethylaminopropyl) carbodiimide at pH 5 followed by adjustment to pH 8 and coupling with rabbit IgG resulted in the formation of hippuric acid-IgG conjugates with less than 10% intra- and intermolecular IgG crosslinking. More than 93% of the bonds linking hippuric acid to IgG were stable to hydroxylamine hydrolysis, indicating the peptide properties of these bonds. This two-step process permitted a defined number of hippuric acid moieties to be loaded onto a single IgG molecule and should provide a useful method for the conjugation of molecules containing carboxyl groups to amino groups on a variety of polypeptides.     

Site specificity of metallic ion binding in Escherichia coli K-12 lipopolysaccharide

The site specificity of metallic ion binding in Escherichia coli K-12 lipopolysaccharide was assessed by collecting high-resolution phosphorus nuclear magnetic resonance spectra in the presence of manganese, a paramagnetic divalent cation. This technique revealed high-affinity interactions between the cation and all of the lipopolysaccharide phosphoryl groups. To ascertain whether the carboxyl groups of 2-keto-3-deoxyoctonate contributed to the metal cation binding, lipopolysaccharide was chemically modified using a glycine ethyl ester - carbodiimide reaction. Of the three available carboxyl groups, only one was neutralized by the exogenously added ligand; the others appeared to be cross-linked within the molecule. By analogy, only one carboxyl group should be freely available for binding metallic ions, while the others are probably neutralized by the close proximity of endogenous amino substituents. Although high-resolution phosphorus nuclear magnetic resonance showed that an intermolecular conformational change had occurred after the carboxyl groups were neutralized, titration with manganese revealed no differences in the apparent strength of the interactions between the cation and the phosphoryl groups. Together, these data suggest that the high affinity of lipopolysaccharide for divalent metallic ions can be attributed primarily to the phosphoryl substituents and not free carboxyl groups.     

Chemical modification of horseradish peroxidase. Preparation and characterization of tracer enzymes with different isoelectric points.

Horseradish peroxidase (HRP), a plant glycoprotein with a molecular weight of 40,000 D and a molecular radius (ae) of 30 A, has been modified chemically to prepare tracer molecules with different molecular charge. Modification of free carboxyl groups on the enzyme is achieved by carbodiimide activation and subsequent reaction of activated carboxyl groups with a nucleophile; uncharged groups or radicals containing additional positively charged moieties are introduced into the protein molecule resulting in an increased net positive charge of the tracer. Amino groups in the protein molecule are modified by acetylation or succinylation; this reaction will increase the net negative charge of the enzyme by either introducing an uncharged group or an additional carboxyl radical. The tracer molecules so obtained are then characterized in terms of molecular size and charge by column chromatography and isoelectric focusing respectively. The enzymatic activity as measured by 3,3'-diaminobenzidine reaction, the pH optimum and the absorption spectra for the modified enzymes remain virtually unchanged.     

Chemical modification of α-galactosidase from coconut

α-Galactosidase from coconut kernel was inhibited by chemical modification of its tyrosine, tryptophan and carboxyl groups. Treatment with N-bromosuccinamide and tetranitromethane indicated that modification of one tryptophan and one tyrosine residue inhibited enzyme activity by 55 and 84%, respectively. Modification of carboxyl groups by carbodiimide indicated that inhibition was due to modification of two carboxyl groups. In the presence of the competitive inhibitor D-galactose, α-galactosidase was protected from inhibition by N-bromosuccinamide, tetranitromethane and carbodiimide. These results indicate that a tryptophan, tyrosine and two carboxyl groups are at or near the active site of α-galactosidase.     

Determination of the cross-linking effect of adipic acid dihydrazide on glycoconjugate preparation

The cross-linking effect of adipic acid dihydrazide (ADH) on polysaccharide derivatization can be evaluated by applying combination of elemental analysis and colorimetric assay. Elemental analysis is used for estimation of total ADH bound to polysaccharide and a colorimetric trinitrobenzene sulfonic acid assay is used to determine the part of ADH not involved in cross-linking. The difference of values expressed as molar ratios (per repeating unit) provides information on the amount of ADH involved in cross-linking the polysaccharides. Carboxymethylated polysaccharides were derivatized with different amounts of ADH to test the procedure. Analytical results showed that excess of ADH in the reaction only slightly decreased the cross-linking. The number of carboxyl groups remained unmodified even at high excess of ADH and high concentration of carbodiimide (EDC) coupling reagent.     

Carbodiimide inactivation of Na,K-ATPase. A consequence of internal cross-linking and not carboxyl group modification

Irreversible inhibition of Na,K-ATPase and K+-dependent p-nitrophenylphosphatase activities was produced by incubation of purified Na,K-ATPase enzyme with 1-ethyl-3(3-dimethylaminopropyl)carbodiimide (EPC). Inhibition was time and [EPC] dependent and displayed first order kinetics with respect to time. The [EPC] to reduce the enzyme velocity by 50% for Na,K-ATPase and phosphatase activities was 1.6 and 2.2 mM, respectively. Analysis of the kinetics of inhibition by EPC indicated that reaction at one site was sufficient to produce inhibition. Inhibition was greatly reduced by the presence of Mg2+, Na+, K+, choline, or Tris (decreasing order of effectiveness); ATP was without effect. This suggests that cation-bound enzyme forms were less reactive with the carbodiimide than free enzyme; ATP-bound enzyme was as reactive. Apparently the cations Na+, Mg2+, Tris, and choline stabilize E1 forms of the enzyme which are different from the E1 form stabilized by ATP. Addition of [14C]glycine ethyl ester (Gly-OEt) resulted in incorporation of radioactivity into both alpha and beta subunits that was dependent upon the presence of EPC, and the incorporation was reduced by the cations which reduced the inhibition due to EPC. Simultaneous addition of Gly-OEt with EPC prevented inhibition, although 14C incorporation still took place. If Gly-OEt addition was delayed the initial inactivation was not affected, but little subsequent inactivation occurred. The protection against inactivation by EPC occurs on the addition of other exogenous nucleophiles, such as aminoethane or ethylenediamine. Dicyclohexylcarbodiimide, a more potent hydrophobic carbodiimide inhibitor, shows similar effects; the inhibition due to dicyclohexylcarbodiimide is also prevented by the simultaneous presence of a nucleophile. After treatment with a carbodiimide and exogenous nucleophile the Na,K-ATPase has modified carboxyl residues but is not inhibited. Thus, modification of the cation-protectable carboxyl groups does not by itself cause inhibition. It seems likely that the inhibition of activity due to carbodiimide alone is not due to the modification of a carboxyl group per se but to the formation of an intramolecular bond between the carbodiimide-activated carboxylic acid and an endogenous nucleophile. The formation of such bonds suggests the close juxtaposition of amine and carboxyl groups in the secondary structure of the enzyme.     

A fluorescent water soluable carbodiimide-2-hydroxy-3-naphthoic acid hydrozide reaction for the demonstration of carboxyl groups in proteins and mucosubstances.

The carbodiimide-2-hydroxy-3-haphthoic acid hydrazide reaction as developed by Geyer (1964) was used without subsequent diazonium coupling as a fluorescent method for the demonstration of carboxyl groups in both proteins and mucosubstances. The topological distribution of the fluorophore was similar to that reported by Geyer. Quantitative microfluorometric studies on cartilage sections revealed differences in detail between emissions in cartilage matrix mucoprotein as compared to the dense connective tissue associated with the perichondrium which consists principally of protein. It would also appear that the primary fluorescent emission of unstained preparations at 450 mm should be useful in microfluorometric determinations of proteins.     

The chemical modification of papain with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide.

The reaction of the water-soluble carbodimide, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), with active papain in the presence of the nucleophile ethyl glycinate results in an irreversible inactivation of the enzyme. This inactivation is accompanied by the derivatization of the catalytically essential thiol group of the enzyme (Cys-25) and by the modification of 6 out of 14 of papain's carboxyl groups and up to 9 out of 19 of the enyzme's tyrosyl residues. No apparent irreversible modification of histidine residues is observed. Mercuripapain is also irreversibly inactivated by EDC/ethyl glycinate, again with the concomitant modification of 6 carboxyl groups, up to 10 tyrosyl residues, and no histidine residues; but in this case there is no thiol derivatization. Treatment of either modified native papain or modified mercuripapain with hydroxylamine results in the complete regeneration of free tyrosyl residues but does not restore any activity. The competitive inhibitor benzamidoacetonitrile substantially protects native papain against inactivation and against the derivatization of the essential thiol group as well as 2 of the 6 otherwise accessible carboxyl groups. The inhibitor has no effect upon tyrosyl modification. These findings are discussed in the context of a possible catalytic role for a carboxyl group in the active site of papain.