Amines with Glutaraldehyde

Amino groups on proteins may be reacted with the bis-aldehyde compound glutaraldehyde to form aaivated derivatives able to cross-link with other proteins. The reaction mechanism for this modification proceeds by one of several possible routes. In the first option, one of the aldehyde ends can form a Schiff base linkage with e-amines 

Schiff base interactions between aldehydes and amines typically are not stable enough to form irreversible linkages. These bonds may be reduced with sodium cya-noborohydride or a number of other suitable reductants (Chapter 3, Section 4) to form permanent secondary amine bonds. Fiowever, proteins cross-linked by glutaraldehyde without reduction nevertheless show stabilities unexplainable by simple Schiff base 

Reductive Amination to Secorxlary Amine Linkage with Terminal Formyl Group 

Glutaraldehyde also can be used to create aldehydes on amine-containing polymers. The use of this reagent in derivatizing chromatography supports and other soluble polymers is well known (Hermanson etal., 1992). 

Dissolve the protein or other amine-containing macromolecule to be modified at a concentration of 1-10 mg/ml in a buffer having a pH from 7 to 10. The higher the pH, the more efficiently Schiff base formation will occur. Phosphate, borate, and carbonate buffers at 0.01—0.1 M are acceptable. Avoid amine-containing buffers like Tris and glycine, since they will react with glutaraldehyde. 

Dissolve the protein or other amine-containing macromolecule to be modified at a concentration of l-10mg/ml in a buffer having a pH from 7 to 10. The higher the pH, the 

Add a quantity of glutaraldehyde equal to a 10-fold molar excess over the amount of amines to be modified. A typical concentration of glutaraldehyde in the reaction mixture is 1.25 percent. In some cases, trial experiments will have to be done to check for solubility of the resultant modified protein. Scale back the quantity of glutaraldehyde added if precipitation occurs. 

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Fig. 17 (a) Elastin-based stimulus-responsive gold nanoparticles. Reproduced from [131] by permission of The Royal Society of Chemistry (b) Functionalization of a glass surface with ELP. In the first step, the glass surface is aminosilylated with N-2-(aminoethyl)-3-aminopropyl-trimethoxysilane, then modified with glutaraldehyde. Subsequently, the stimulus-responsive biopolymer is covalently immobilized using reductive amination. Reproduced from [132] by permission of The Royal Society of Chemistry... 

Reaction of 1-nitropropane with glutaraldehyde in aqueous ethanol in the presence of sodium hydroxide yields a mixture of two products, the major component of which, lr-ethyl-l-nitrocyclohexane-2c,6f-diol (98), can be isolated in 36% yield ). Acid-catalyzed acetylation converts (98) into the di-O-acetate, hydrogenation yields the corresponding amine, which has been characterized as the hydroacetate, N-acetate and triacetate. Configurational assignments followed from NMR data, which clearly showed the steric non-equivalence of the two hydroxyl groups vicinal to the tertiary center. 

Branched-chain L-amino acids can be analyzed using leucine dehydrogenase immobilized on aminated PVA activated with glutaraldehyde. This enzyme catalyzes the deamination of L-Leu, L-Ile, and L-Val to 2-oxo acids in the presence of NAD. ... 

Aromatic amines are activators of horseradish peroxidase. Kulys and Vidziunaite (1983) adsorbed HRP together with GOD on a carbon electrode and crosslinked the enzymes with glutaraldehyde to assemble a sensor for aromatic amines. H2O2 is produced in the presence of glucose, acting as cosubstrate in the HRP-catalyzed oxidation of ferrocyanide. The ferricyanide formed was reduced back to ferrocyanide at an electrode potential of +10 mV vs SCE, the current being limited by the... 

Enzymes have also been immobilized on collagen membrane after its stepwise modification to esters, hydrazides and azides [171]. Another method of enzyme electrode preparation consists of enzyme immobilization on polyacryl acid modified with p-nitroaniline and by a subsequent reduction of N02-groups with titanous chloride and following diazota-tion of resulting aromatic amines [150]. An enzyme electrode has also been prepared by the direct immobilization of an enzyme on the surface of a Pt-electrode which was formerly modified first with 3-aminopropyl triethoxysilane and secondly with glutaraldehyde and bovine serum albumin [172]. Enzymes can also be immobilized on p-benzoquinone-carbon paste[173] or on the graphite electrode after its activation with cyanuric chloride [174]. In a similar way an enzyme electrode has been prepared by using iridium diiodide electrode as a support [175]. 

Mechanism of action. Chemically, glutaraldehyde is 1,5-pentanedial. As an aldehyde, it is a reactive molecule that undergoes those chemical reactions that are typical of any aldehyde. Its applications as a microbicide rely on the fact that it reacts with the free amine (non-protonated) form of primary and secondary amines in an irreversible manner. The pH of the surrounding medium, along with the pKa of the amine, will determine whether an amine is in its protonated or non-protonated (free) form. In acidic solutions, amines are generally protonated and their reaction with glutaraldehyde are slower than in alkaline systems where the amine is not protonated. For this reason, glutaraldehyde works faster in alkaline systems than acidic systems. 

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