Art Labeling Activity Figure 54 2 of 2 Bone Matrix

Introduction

Among the many available protein crosslinking agents, glutaraldehyde has undoubtedly found the widest application in various fields such as histochemistry (ane–three), microscopy (1,four,five), cytochemistry (6), leather tanning industry (7,viii), enzyme technology (9–13), chemical sterilization (fourteen), and biomedical (15) and pharmaceutical sciences (16). Glutaraldehyde, a linear, 5-carbon dialdehyde, is a clear, colorless to pale straw-colored, pungent oily liquid that is soluble in all proportions in water and booze, as well as in organic solvents. It is mainly available every bit acidic aqueous solutions (pH 3.0–iv.0), ranging in concentration from less than 2% to 70% (due west/five). Glutaraldehyde has had great success because of its commercial availability and depression cost in addition to its high reactivity. It reacts apace with amine groups at effectually neutral pH (17) and is more efficient than other aldehydes in generating thermally and chemically stable crosslinks (15). In fact, studies of collagen crosslinking reactions with monoaldehyde (formaldehyde) and dialdehydes having concatenation lengths of two to six carbon atoms (glyoxal, malonaldehyde, succinaldehyde, glutaraldehyde, and adipaldehyde) demonstrated that the reactivity in this series is maximized at five carbons; thus glutaraldehyde is the most constructive crosslinking amanuensis (18).

Glutaraldehyde has institute widespread use for enzyme immobilization. Despite the success of this reagent, its chemical science has been quite controversial (19). In fact, the simple structure of glutaraldehyde is non indicative of the complication of its behavior in aqueous solution and its reactivity. Our purpose here is to review the literature on glutaraldehyde past first presenting its chemical behavior in aqueous solution and and so its reactivity with proteins, focusing on its awarding for enzyme immobilization.

Chemic Behavior of Glutaraldehyde in Aqueous Solution

Knowledge of the structure and machinery of crosslinking reagents is important for their use. However, the structure of glutaraldehyde in aqueous solution has been the discipline of more debate than whatever of the other crosslinking reagents. In fact, glutaraldehyde structure in aqueous solution is non limited to the monomeric grade (Figure ane, structure I). Figure 1 gives an overview of the possible molecular forms of glutaraldehyde in aqueous solution based on reports covering the by 40 years (ten,20–29). The dissimilar glutaraldehyde structures (Figure one) accept been numbered as found in the literature and are discussed in chronological social club.

Figure 1. Summary of the possible forms of glutaraldehyde in aqueous solution.

In 1962, Aso and Aito (20,30) studied the polymerization of glutaraldehyde using cationic catalysts, and they establish that a like polymerization occurred spontaneously in aqueous solutions, at room temperature in the absenteeism of a catalyst. The last product (structure 8) was proposed to be a soluble tetramer or pentamer containing approximately one free aldehyde grouping per molecule formed through the intra-molecular-intermolecular propagation polymerization with ring formation.

In 1968, Richards and Knowles (10) studied glutaraldehyde solutions by proton (H) nuclear magnetic resonance (H-NMR). The NMR data (i.e., types of protons and peak integrations) did not concur with those expected from dimer, cyclic dimer, trimer, or bicyclic trimer simply, merely rather was consistent with a mixture of polymeric forms of these oligomers likewise as college polymeric species. The authors ended that commercial solutions were largely polymeric and contained meaning amounts of α,β-unsaturated aldehydes (structure Six) that were able to course rings (structure VII) by loss of water molecules past aldol condensation. The structure VI represents the average structure of the unsaturated polymerized glutaraldehyde (α,β-unsaturated compound), and Hooper (31) reported that the pendent aldehyde groups of construction 6 would be scarcely hydrated because the carbonyl course is stabilized by conjugation.

Hardy et al. (21), in 1969, used ultraviolet (UV) spectrophotometry in combination with H-NMR to investigate glutaraldehyde solutions. They too institute α,β-unsaturated aldehydes (structure Vi), simply only equally a very minor component of the organic content because of the relatively weak absorbance observed at 235 nm. Furthermore, after purification of glutaraldehyde by liquid extraction with ethyl ether, these authors obtained 50% pure glutaraldehyde (structure I) with the expected H-NMR spectrum. Additional H-NMR investigations showed that purified glutaraldehyde undergoes very rapid hydration upon dissolution in water, which agreed with the results of Aso and Aito (20,thirty). Thus, Hardy et al. (21) postulated that glutaraldehyde monomer (structure I) existed as a mixture of hydrated forms (structures 2, Iii, and Iv) in aqueous solution, all of them beingness in equilibria.

In their work a few years after, Korn et al. (22) did not notice meaning amounts of α,β-unsaturated aldehydes (structure VI) by UV-spectrophotometric analysis, which was in understanding with the findings of Hardy et al. (21). They proposed, after H-NMR analysis, that aqueous solutions of glutaraldehyde consisted of complimentary glutaraldehyde (structure I), the cyclic hemiacetal of its hydrate (structure 4), and oligomers of this (construction V), all in equilibria and in different proportions as a function of the temperature. In 1974, Whipple and Ruta (32) studied aqueous glutaraldehyde using 13C-NMR and concluded that aqueous glutaraldehyde consisted primarily of the circadian hemiacetal (construction IV), merely they added that this class was as distributed between two geometrical isomeric forms (cis and trans).

Monsan et al. (23), in 1975, confirmed, for acidic glutaraldehyde solutions, the results obtained by Hardy et al. (21), Korn et al. (22), and Whipple and Ruta (32) using gel and thin-layer chromatography, mass spectrometry (MS), H-NMR, and infrared spectroscopy (IR). For glutaraldehyde solutions at neutral or slightly alkali metal pH, they observed the production of molecular forms, which could precipitate. They also noted the formation of an abundant precipitate, named poly-glutaraldehyde, when sodium hydroxide was added to 25% aqueous glutaraldehyde solution to achieve pH 11.0. MS and IR analysis immune the identification of the solid as being the issue of aldolic condensation of glutaraldehyde molecules (structures VI and/or VII).

In 1980, Margel and Rembaum (25) investigated the aldol condensation of glutaraldehyde in the pH range of 7.0 to thirteen.five. They obtained poly-glutaraldehyde and proposed the structure 9 after spectroscopic and electrochemical analysis. They besides reported that poly-glutaraldehyde may be soluble or insoluble in water and may have different concentrations of aldehyde, hydroxyl, and carboxylic functional groups depending on the polymerization weather condition, such as pH and oxygen content, leading to the structures XII and 13.

In 1991, Tashima et al. (27) obtained a new dimer when glutaraldehyde was treated by aqueous alkaline solution (pH 8.five). Their analyses by UV and IR suggested the existence of α,β-unsaturated formyl and hydroxyl groups in this molecule, and gas chromatography (GC) MS assay indicated the molecular formula CxHfourteenO3 (molecular weight: 182 m/mol). Moreover, after derivatization followed by ii-dimensional NMR analysis, they proposed the structure X, in equilibrium with structure XI, for the glutaraldehyde dimer.

In 1992, Kawahara et al. (28) reported that most of the studies summarized above (x,21,23,32) neglected possible solvent effects on the glutaraldehyde structure. In fact, h2o is the medium in which commercial glutaraldehyde is supplied and in which the crosslinking reaction with proteins is carried out, and glutaraldehyde was found to react with this solvent in various ways. Thus, according to Kawahara et al. (28), there was a considerable problem in the studies carried out by Monsan et al. (23) because their analyses were conducted only in organic solvent (tetrahydrofuran for gel chromatography, chloroform/acetone for thin-layer chromatography, and deuterated chloroform or carbon tetrachloride for H-NMR). In fact, the equilibrium betwixt monomeric and polymeric glutaraldehyde in anhydrous solvents could perchance shift to the latter to produce water. Other studies showed like issues (10,21,32). For example, Richards and Knowles (10) and Hardy et al. (21) conducted their experiments in deuterated h2o, just the commutation of deuterium for hydrogen bound to the α-carbon could give erroneous results when comparing the peak intensities by H-NMR (22). Moreover, the hydration equilibrium constants for monoaldehyde (formaldehyde) are reported to differ in water and deuterated water (33), and this probably occurs with glutaraldehyde. Whipple and Ruta (32) used 13C-NMR to analyze glutaraldehyde solutions, but direct comparing of the peak intensities is known to be not quantitative in 13C-NMR (34). Thus, these observations led Kawahara et al. (28) to investigate the molecular construction of glutaraldehyde in aqueous solution by UV absorption and light handful. The 70% (west/v) glutaraldehyde solution used for their written report was plant to contain a large quantity of polymeric species with circadian hemiacetal structure (V). Upon dilution, the polymerized glutaraldehyde slowly converted to monomers, thus inducing a corking variation in the relative abundances of monomeric and polymeric species, according to the glutaraldehyde concentration. In fact, they institute that in dilute solution and in the pH range of iii.0 to viii.0, glutaraldehyde was almost monomeric, predominantly in cyclic hemiacetal course (structure 4). In 1997, the same authors (29) establish that glutaraldehyde structure was similar for aqueous solutions up to 10% (w/v) and ended that the content of α,β-unsaturated structures (structure VI) was negligible regardless of the glutaraldehyde concentration.

In summary, several studies (ten,20–29) have shown that commercially available glutaraldehyde represents multicomponent mixtures, only knowing which of these components is the nigh efficient for reactions with proteins is debatable. In fact, in aqueous solution, glutaraldehyde can be in its simplest form, a monomeric dialdehyde, only also as a dimer, trimer, and polymer. Therefore, the effectiveness of glutaraldehyde immobilization and the controversies surrounding its chemical behavior could exist rationalized with the multiplicity of structures, which depends on the solution weather.

Application to Substances of Biological Interest

Carbohydrates, Lipids, and Nucleic Acids

There is little information on the use of aldehydes to set carbohydrates (35) or lipids (6). Most lipids practise not react well with glutaraldehyde, with the exception of some phospholipids that comprise principal amines (e.g., phosphatidylserine and phosphatidylethanolamine; Reference 36). In the instance of nucleic acids, formaldehyde is past far the nearly effective agent for their fixation by reaction with amino groups of DNA nucleotides (37), but little information is available on the interaction between glutaraldehyde and DNA (38).

Proteins: General Instance

Glutaraldehyde was used for the first fourth dimension at the outset of the 1960s for the fixation of tissues (1), and since this time many other applications have been developed. The high reactivity of glutaraldehyde toward proteins at around neutral pH is based on the presence of several reactive residues in proteins and molecular forms of glutaraldehyde in aqueous solution, leading to many different possible reaction mechanisms. Enzyme immobilization represents a practiced instance to illustrate the use of glutaraldehyde as protein crosslinking reagent.

Glutaraldehyde tin react with several functional groups of proteins, such as amine, thiol, phenol, and imidazole (39) because the most reactive amino acid side-bondage are nucleophiles. Various data on aldehyde reactivity (at pH from 2.0 to 11.0) with the following amino acids have been reported in the literature: lysine (18); tyrosine, tryptophan, and phenylalanine (forty); histi-dine, cysteine, proline, serine, glycine, glycylglycine, and arginine (41). These authors investigated the ability of different aldehydes to react with amino acids, and they ranked the reactive moieties of the amino acids in decreasing order of reactivity equally follows: ϵ-amino, α-amino, guanidinyl, secondary amino, and hydroxyl groups. Avrameas and Ternynck (42) ended that either glutaraldehyde did not react with the amine function of the guanidinyl group (arginine) or that in protein molecules the more reactive groups prevented the ascertainment of arginine reactivity with glutaraldehyde. Okuda et al. (17) noted that glutaraldehyde reacted with thiol groups only in the presence of a primary amino group. Glutaraldehyde reacts reversibly with amino groups over a wide pH range (≥pH three.0), except between pH vii.0 to 9.0 where only a lilliputian reversibility is observed (17).

The crosslinking of proteins, either to a carrier (solid back up) or betwixt poly peptide molecules (carrier-free), generally implies the ϵ-amino grouping of lysyl residues (43). The unprotonated amino groups are very reactive as nucleophilic agents (44). It should be noted that lysyl ϵ-amino groups take pKa (acid dissociation abiding) > 9.5, but information technology is presumed that the small percentage of amines nowadays in their unprotonated form at lower pH is sufficient to react with glutaraldehyde, which then drives the acid-base equilibrium to deprotonation of these groups for further reaction. Nearly proteins contain many lysine residues, usually located on the protein surface (i.e., exposed to the aqueous medium) because of the polarity of the amine group. Furthermore, lysine residues are generally not involved in the catalytic site, which allows moderate crosslinking to preserve protein conformation (45) and thus biological activeness (46). As stated previously, glutaraldehyde exists in multiple forms in aqueous solution, and all of these forms might be reactive toward lysine residues (ϵ-amino group) of proteins.

In spite of the substantial corporeality of literature concerning the use of glutaraldehyde, at that place is still no agreement most the main reactive species in glutaraldehyde solutions during the cross-linking process. Aldehydes are expected to form Schiff bases upon nucleophilic attack past the ϵ-amino groups of lysine residues in the protein (23). All the same, Schiff bases are unstable under acidic weather condition and tend to break downward to regenerate the aldehyde and amine. In contrast, the linkage formed past the reaction of glutaraldehyde with an amino group has shown exceptional stability at extreme pHs and temperatures, thus a simple Schiff base with both ends of monomeric glutaraldehyde has been ruled out as a mechanism for glutaraldehyde crosslinking with proteins. Several alternative mechanisms have been proposed.

Betwixt 1968 and 1975, Richards and Knowles (10) and Monsan et al. (23) postulated pathways, both involving the reaction of the protein amino group with α,β-unsaturated aldehydes formed by aldol condensation of glutaraldehyde (Effigy ii). Richards and Knowles (ten) proposed that the reaction involved the conjugate add-on of protein amino groups to ethylenic double bonds (Michael-type add-on) of the α,β-unsaturated oligomers found in the commercial aqueous solutions of glutaraldehyde that are usually used (Figure 2, reaction 2). A few years afterward, Peters and Richards (47) showed work that supported this hypothesis considering they establish that, in the presence of an excess of amino groups, nucleophilic improver on the ethylenic double bail was possible. Monsan et al. (23) proposed a slightly different mechanism in which an addition reaction occurred on the aldehydic role of the α,β-unsaturated polymers (and poly-glutaraldehyde) to give a Schiff base of operations (imine) stabilized by conjugation (Figure 2, reaction 1).

Figure 2. Schiff base (ane) and Michael-type (2) reactions of glutaraldehyde with proteins.

In the early 1970s, Boucher (48,49) proposed that monomeric glutaraldehyde was the agile species involved in the crosslinking with proteins and that the facility of polymeric forms to revert to the active monomer depended upon pH (i.e., the type of glutaraldehyde polymers in solution). He also considered that polymers existing in the alkaline pH range cannot revert to the monomer because time and temperature tend to favor a more irreversible polymer, in dissimilarity to polymers that exist at acidic and neutral pH. This was supported in 1990 when Ruijgrok et al. (50) showed that glutaraldehyde polymers in the neutral and acidic pH range could revert easily to the monomer under the influence of heating or ultrasonic radiation.

As early on equally 1976, Hardy et al. (51,52) and Lubig et al. (53) argued that the reaction of glutaraldehyde with proteins was not due to α,β-unsaturated aldehydes but may involve some dimerization in the presence of the amino grouping, such as the formation of quaternary pyridinium compounds (Figure 3), rather than glutaraldehyde polymers reacting with amino groups. The machinery involved could result from cyclization, dehydration, and internal redox reactions of a Schiff base. Hardy et al. (52) reported the isolation of a pyridinium-blazon chemical compound following the reaction of glutaraldehyde with amines and suggested this structure every bit a stable crosslink. They showed that this compound had an UV assimilation maximum at 265 nm, which was consistent with the original observation of Bowes and Cater (18).

Figure iii. Crosslinking of proteins with glutaraldehyde giving a fourth pyridinium compound.

In 1991, Tashima et al. (27) concluded that reaction of alkaline metal glutaraldehyde solutions (mixture of dimers 10 and Xi) with proteins may involve a Michael addition to the double bonds to give (Xa) and (XIa), as shown in Effigy 4 (adapted from Reference (19). If these reactions occur, no reduction is necessary to stabilize the adducts. If an backlog of amine is added, the compound (XIb) may be formed.

Figure 4. Reaction of dimeric circadian glutaraldehyde with proteins nether basic conditions.

In 1994, Walt and Agayn (19) proposed multiple reaction products for the dissimilar glutaraldehyde structures in solution depending on the pH conditions because each course of glutaraldehyde might participate differently in crosslinking reactions with proteins. Thus, under acidic or neutral conditions, glutaraldehyde exists equally a mixture of monomers [i.east., free aldehyde form (I) or cyclic hemiacetal (IV)] or as a polymer [i.e., cyclic hemiacetal oligomer (5)]. Each of these structures would exist expected to form Schiff bases upon nucleophilic attack past lysine residues in a protein, as shown in Figure five. However, every bit previously mentioned, Schiff bases are unstable under acidic conditions and thus Schiff base of operations formation (Figure 5, Equation ane) between a lysine amino grouping and free aldehyde (structure I) is not favored. Information technology is more likely that monomeric circadian hemiacetal (IV) and its multimeric form (V) react via reactions in Equations 2 and 3 of Effigy 5, under acidic conditions. Under basic conditions (Figure 6), the reaction of α,β-unsaturated oligomeric aldehydes (VI) with amine can give two products robust to acid hydrolysis: a Schiff base of operations (Effigy 6, structure VIa), which was more stable because of the conjugation of the internal aldehyde grouping with the C-C double bonds and a Michael improver product (VIb). In the presence of excess amine, a mixed product (VIc) is seen, which is labile to acrid hydrolysis considering of the disruption of resonance stabilization. Because elimination of water in the formation of Schiff bases is reversible and because prolonged exposure to buffer solutions, particularly at elevated pH, might impair bounden and lead to gradual release of the enzyme, reducing the double bonds of the Schiff bases by application of suitable reducing agents (19) such as sodium borohydride (NaBH4) or sodium cyanoborohydride (NaCNBH3) has been proposed. In both cases, the reduction of (VIa) produces a secondary amine that is tolerant to variations in pH and is stable fifty-fifty in acidic conditions. Sodium cyanoborohydride is preferred because it is a milder reagent (54). In fact, sodium borohydride not only reduces Schiff bases, but also aldehyde groups, leading to a lower yield of conjugate formation (55). Even if the employ of a reducing amanuensis has been recommended, reduction is non usually required (19).

Figure 5. Reactions of glutaraldehyde with proteins nether acidic or neutral conditions.
Figure 6. Reaction of polymeric glutaraldehyde with proteins nether basic conditions.

In 1997, Kawahara et al. (29) speculated on whether proteins could catalyze the aldol condensation/polymerization of glutaraldehyde. They suggested that monomeric glutaraldehyde could exist converted to polymeric forms by the action of amino groups and that this product played a major role in the cantankerous-linking reaction of proteins. In fact, they proposed that the polymerization of glutaraldehyde via aldol condensation proceeded in parallel with the cross-linking reaction and that the formation of a Schiff base (imine) past one glutaraldehyde molecule with one amino group enhances its aldol condensation with other glutaraldehyde molecules. The final crosslinked structure would be a linear aldol-condensed oligomer of glutaraldehyde, with several Schiff base linkages branching off. They also observed that the dehydration step following aldol condensation occurs almost completely at the glutaraldehyde monomer units containing Schiff base imine, in contrast to the glutaraldehyde units containing no Schiff base of operations, where little dehydration occurs. Therefore, the formed Schiff base linkage eventually constitutes a conjugate system with the adjacent ethylenic double bond. Once such conjugation is formed, the resonance interaction is reported to make Schiff base linkages stable to acrid hydrolysis (23).

In conclusion, the chemical nature of the reaction of glutaraldehyde with proteins is non conspicuously understood, and the mechanisms of protein crosslinking reactions remain open up to speculation. However, information technology seems that no single machinery is responsible for glutaraldehyde reaction with proteins. In fact, considering glutaraldehyde is present in dissimilar forms fifty-fifty for specific and controlled reaction weather, several of the possible reaction mechanisms presented above could go along simultaneously.

Enzymes

Immobilized enzymes are currently the subject of considerable involvement because of their advantages over soluble enzymes or alternative technologies, and their applications are steadily increasing. Immobilization by covalent attachment to water-insoluble carriers via glutaraldehyde is one of the simplest and near gentle coupling methods in enzyme technology (43). The showtime reported application of the apply of a bifunctional reagent was by Zahn in the 1950s (56), which was followed past studies on the chemistry of crosslinking with glutaraldehyde for the grooming of stable protein crystals for X-ray diffraction studies (45) or for the fixation of tissue samples for microscopic investigation (57). Later, glutaraldehyde was widely used every bit a mild cross-linking agent for the immobilization of enzymes because the reaction proceeds in aqueous buffer solution under conditions close to physiological pH, ionic strength, and temperature. Essentially, ii methods have been used: (i) the formation of a three-dimensional network as a result of intermolecular crosslinking and (ii) the bounden to an insoluble carrier (eastward.k., nylon, fused silica, controlled pore drinking glass, crosslinked proteins such every bit gelatin and bovine serum albumin (BSA), and polymers with pendant amino groups).

Immobilization can be achieved for many enzymes under a wide range of conditions, which should be chosen according to the specific results required. These weather have often been adamant past trial and mistake because insolubilization is critically dependent on a delicate residuum of factors such every bit the nature of the enzyme (42,58,59), the concentration of both enzyme (sixty) and reagent (58), the pH (61) and ionic strength (62) of the solution, the temperature (63), and the reaction time (64).

The nature of the enzyme, particularly its lysine content, has an effect on its insolubilization past glutaraldehyde (42,58,59). Moreover, if simply a small amount of enzyme is available or if extensive modification must be avoided, the addition of inert, lysine-rich protein (e.thousand., BSA) has been suggested by Broun (58).

As mentioned above, the concentrations of enzyme and glutaraldehyde must be carefully considered to obtain water-insoluble enzyme derivatives via crosslinking (lx); depression concentrations of enzyme and glutaraldehyde tend to induce intramolecular crosslinking by enhancing the probability that glutaraldehyde functional groups will react with the same enzyme molecule (threescore). Thus, conditions should be called advisedly to favor intermolecular crosslinking between enzyme molecules instead of unwanted intramolecular links, which could also be formed (58,65,66). Broun (58) reported that the amount of crosslinking amanuensis used affects the degree or extent of crosslinking. He indicated that low concentrations of glutaraldehyde were not able to form sufficient crosslinkages to effect atmospheric precipitation of the enzyme. At higher concentrations, the extent of crosslinking was loftier enough to grade a tight construction by excluding water molecules to insolubilize the enzyme derivative. Chui and Wan (67) indicated that enzymatic activity was inversely proportional to the concentration of glutaraldehyde used because extensive crosslinking may outcome in a distortion of the enzyme structure (i.e., the active site conformation). With this distortion, the accessibility and accommodation of the substrate may be reduced, thus affecting the retentiveness of biological activity. Furthermore, the relative concentration of enzyme to glutaraldehyde should besides be considered (17). Nosotros institute that crosslinking of the enzyme trypsin (EC iii.iv.21.4) with glutaraldehyde could exist accomplished over a broad range of relative mole ratios in l mM sodium phosphate buffer at pH 6.8 merely that the time required to commence precipitation ranged from 0.5 to 120 min for enzyme:glutaraldehyde ratios of 1:500 to 1:25, respectively (I. Migneault, unpublished data).

The reaction of glutaraldehyde with enzymes to give soluble and insoluble products has been extensively studied, and the reaction was shown to be pH-dependent (39). Jansen et al. (61) showed that the optimum pH for glutaraldehyde insolubilization varied from protein to protein. In fact, they observed that the pH values for the almost rapid insolubilization of BSA, soybean trypsin inhibitor, lysozyme (EC iii.two.1.17), and papain (EC iii.iv.22.ii) were plant to be nearly the aforementioned as the isoelectric points (pIs) of these proteins, whereas the formation of insoluble active chymotrypsin (EC three.4.21.1) was almost rapid at pH 6.2 (pI 8.6) and chymotrypsinogen A at pH 8.2 (pI 9.five). The beingness of an optimum pH suggests the important role of the protein accuse on the intermolecular crosslinking required for insolubilization. The accuse on the poly peptide may regulate crosslinking, which was maximal when the repulsive charges were minimal. Furthermore, Tomimatsu et al. (62) and Broun (58) concluded that the lower the ionic strength of the reaction medium, the more rapid the crosslinking of chymotrypsin. On the other hand, the choice of pH should as well exist taken into account regarding the reactivity of aqueous glutaraldehyde, almost immobilizations existence conducted in the neutral or slightly alkaline metal pH range.

The influence of temperature and reaction time on insolubilization of enzymes has been reported by Broun (58). In early on reports on enzyme immobilization, the reactions were carried out at low temperature (4°C), which was preferred for labile molecules, merely the immobilization process required long reaction times (six–18 h; Reference (59). Ottesen et al. (63) and Bullock (35) suggested that the reaction of glutaraldehyde with lysine residues was progressive with time, probably depending on the accessibility of the ϵ-amino groups. Currently, ambient temperature is used for glutaraldehyde immobilization of enzymes within iv h or less (68,69).

The catalytic activity of water-insoluble enzyme derivatives prepared using multifunctional reagents such every bit glutaraldehyde can vary considerably (61,63,70) and has been shown to be dependent on the amount of crosslinking reagent used during insolubilization, as well as on other factors (71). Moreover, the kinetic behavior of immobilized enzymes is, in many respects, different from that of free enzyme in solution (72), these differences being related to the diverse microenvironments generated past the enzymatic hydrolysis of the substrate. Kinetic properties of soluble enzymes are expressed in terms of Michaelis-Menten parameters. In the case of immobilized enzymes, credible kinetic properties are used because the overall kinetic behavior of the enzymatic preparation is the sum of isolated contributions of each individual enzyme molecule, which tin be immobilized via different amino groups, leading to different exposures of the catalytic centers (73). Our work (74) on trypsin immobilization with glutaraldehyde either by covalent attachment to aminopropyl controlled pore glass (CPG) particles or past crosslinking of trypsin in solution showed an increase in the apparent Michaelis constant, KYard,app (i.e., a decrease in enzyme-substrate affinity) relative to free trypsin, which was more than pronounced for glutaralde-hyde-crosslinked trypsin compared to CPG-trypsin. Thus, co-ordinate to our results, the crosslinking process led to a more than constrained enzyme. Furthermore, the shapes of the pH-activity curves depend on the nature of the products liberated besides every bit on the kinetic parameters. Changes in the specificity of sure immobilized enzymes have been reported. For example, glutamic transaminase (EC 2.half dozen.1.1) crosslinked with glutaraldehyde lost its transaminase activity but was nevertheless able to class complexes with its antibody (75).

Stabilities (thermal, chemical, and mechanical) of h2o-insoluble enzyme derivatives have also been described (46,76). Most notably, thermal stability of immobilized enzymes has been shown to vary from greater down to a lesser extent relative to the native enzyme. The stability of an enzyme (protein) tin typically be increased past crosslinking because intra- and intermolecular crosslinks lead to a more rigid molecule that can resist conformational changes (77). In fact, the covalent bonds created during the crosslinking reaction are stable, fifty-fifty in the presence of substrate or loftier ionic strength solutions (59). Moreover, pH and temperature tin be varied over a wide range without dissolution or deterioration of the cantankerous-linked crystals (78). The crosslinking confers mechanical advantages because fragile crystals become much more than sturdy and robust, and then that there is much less risk of damage during handling, while remaining permeable to dissolved solutes. Amongst enzymes, proteases such as trypsin are of great interest because of their numerous applications in many fields. However, most of the commonly used proteases are marginally stable in their soluble form, the prominent cause of their irreversible inactivation existence autoproteolytic digestion. Therefore, stabilization by immobilization has been the subject of considerable research. For example, nosotros digested denaturated lysozyme using ii immobilized trypsin preparations (enzyme either covalently attached to aminopropyl CPG particles or cross-linked with glutaraldehyde) and did not find autoproteolysis (I. Migneault, C. Dartiguenave, J. Vinh, M.J. Bertrand, and Thou.C. Waldron, submitted data). Moreover, these immobilized trypsin preparations showed excellent digestion reproducibility based on liquid chromatographic and capillary electrophoretic peptide maps. Insoluble trypsin preparations were found to be considerably more stable than native trypsin in the alkaline pH range (79). Glassmeyer and Ogle (80) reported that an insoluble trypsin preparation could exist used repeatedly without loss of activity and could be left standing in pH viii.0 buffer at room temperature for 40 h with but a 9% loss of activity. However, the activity of insoluble trypsin preparations was completely destroyed upon incubation at 100°C in pH 8.0 buffer for fifteen min (80). Walsh et al. (81) described the enhanced chemical stability of crosslinked, monolayered trypsin in the presence of urea, and Habeeb (70) reported a college stability of trypsin crosslinked with glutaraldehyde during continuous utilise for casein digestions.

The storage stability of several water-insoluble enzyme derivatives has been examined because an insoluble enzyme derivative should retain activity for considerable periods of time to be useful. For case, Jansen and Olson (64) reported that papain (EC three.4.22.2) crosslinked with glutaraldehyde showed no detectable subtract in esterase activity after 5 months at 4°C. Glassmeyer and Ogle (80) stored insoluble preparations of trypsin in h2o at 4°C for several months and, in almost cases, at virtually a xv% drop in activity was institute. Silman and Katchalski (79) did not note a marked loss of activity afterwards lyophilization and storage at room temperature. On the opposite, catalase (EC 1.eleven.1.half-dozen) crosslinked with glutaraldehyde showed a decrease of about twenty% in its initial activity after ii weeks at iv°C in aqueous solution, after which no further decrease in action occurred after 5 months of storage (eleven). Thus, storage of immobilized enzyme preparations depends on the nature of the product and should be tested for each case.

The application of chemic cantankerous-linking is multidisciplinary, ranging from basic protein biochemistry to applied biotechnology, engineering, and medicine. Since the 1960s, glutaraldehyde has been used to couple enzyme (and protein) to carriers such equally cellulosic materials (82), aminoalkylsilylated drinking glass (83), polyacrylhydrazide (84), phenalanyl-lysine coated polystyrene beads (85), and polyethyleneimine treated magnetite (86). Immobilized enzymes are also used in biosensors (87), chromatographic packings and detectors (88), online solid-stage reactors (89), and in the field of medical diagnostics and therapy (90).

There is an ongoing interest in carrier-complimentary immobilized enzymes, such as crosslinked enzyme crystals (CLECs; References (45) and (91), crosslinked dissolved enzymes (CLEs; References 64, lxx and 74) and crosslinked enzyme aggregates (CLEAs; Reference 92). CLECs results from the chemical stabilization of the crystalline lattice of enzyme molecules past glutaraldehyde, forming highly concentrated immobilized enzyme particles that can be lyophilized and stored for a long fourth dimension at room temperature. CLEs are obtained past the crosslinking of dissolved enzymes, which leads to enzymes with enhanced thermostability. CLEAs were synthesized in a unproblematic and effective way by physical aggregation of the enzyme penicillin Grand acylase (penicillin amidohydrolase, E.C. iii.5.ane.11) using a precipitant (eastward.1000., tert-butyl booze), followed by chemic crosslinking with glutaraldehyde.

A unlike utilization of glutaraldehyde's crosslinking ability, beginning reported in the late 1970s, is related to the preparation of microspheres for a variety of immunological and therapeutic applications (24). Glutaraldehyde was used for the grooming of micro-spheres from proteins (east.g., gelatin) or alginate considering of its splendid fixative properties. Gelatin microspheres have been widely evaluated every bit drug carriers (93). Unfortunately, gelatin dissolves rather rapidly in aqueous environments, making the use of this polymer difficult for the product of controlled and/or long-term delivery systems (94). Thus, the use of a crosslinking procedure that leads to the germination of nonsoluble networks within the microsphere wall is required to reduce dissolution and premature drug release (95). Sodium alginate microspheres have been crosslinked with glutaraldehyde in the presence of calcium chloride (67), and applications of alginate microspheres accept been reported in medicine (96) as well equally in agronomics (97).

Conclusions

The success of glutaraldehyde as a crosslinking agent is a event of its multicomponent nature, where several forms are present in equilibrium in the reagent solution at a given pH. However, the exact molecular limerick of glutaraldehyde solutions, every bit well as which component is the well-nigh reactive, is debatable despite enough of cognition. Equally a result, the reaction mechanism of glutaraldehyde with protein amino groups is not conspicuously understood, as illustrated by the large number of mechanisms reported in the literature and summarized in this review. No single mechanism seems to exist responsible for glutaraldehyde crosslinking with proteins. All reported forms of glutaraldehyde exhibit the ability to react and crosslink proteins, leading to the formation of a broad range of conjugates. A fairly rigid command of reaction conditions is needed to accomplish efficient insolubilization of each different enzyme due to their structural variability. Nevertheless, the resulting enzyme derivatives generally show good stability and thus can be reused. Although partial enzyme inactivation due to chemical modification is often unavoidable, in most cases plenty catalytic action is retained. More work is needed to provide additional information on the exact structure of these crosslinked products.

Competing Interests Argument

The authors declare no competing interests.

References

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