Glutaraldehyde: Nature of the reagent

Korn, Alfred H.; Feairheller, Stephen H.; Filachoine, E.M.

doi:10.1016/0022-2836(72)90206-9

Cited by 234 publications

(100 citation statements)

References 10 publications

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“…This conversion had been suggested to occur by several researchers (18, 20, 26), but its chemical, physiological and ecological consequences had not been studied in detail. As glutaraldehyde is well known as an potent alkylator, crosslinker, and denaturant of protein and is used to fix protein in histochemical studies (27)(28)(29)(30)(31), we hypothesized that the same reaction might occur in our system. To examine this possibility, we mixed 11 mM oleuropein with 5 units of ␤-glucosidase from sweet almond.…”

Section: Resultsmentioning

confidence: 99%

Enzymatic activation of oleuropein: A protein crosslinker used as a chemical defense in the privet tree

Konno

Hirayama²,

Yasui³

et al. 1999

Proc. Natl. Acad. Sci. U.S.A.

207

174

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Leaves of the privet tree, Ligustrum obtusifolium, contain a large amount of oleuropein, a phenolic secoiridoid glycoside, which is stably kept in a compartment separate from activating enzymes. When the leaf tissue is destroyed by herbivores, enzymes localized in organelles start to activate oleuropein into a very strong protein denaturant that has protein-crosslinking and lysine-decreasing activities. These activities are stronger than ever reported from plant systems and have adverse effects against herbivores by decreasing the nutritive value of dietary protein completely. We report here that strong oleuropein-specific ␤-glucosidase in organelles activates oleuropein by converting the secoiridoid glucoside moiety of oleuropein into a glutaraldehyde-like structure, which is also an ␣,␤-unsaturated aldehyde. Oleuropein activated by ␤-glucosidase had very strong proteindenaturing, protein-crosslinking, and lysine-alkylating activities that are very similar to, but stronger than, those of glutaraldehyde. Aucubin, another iridoid glycoside, had similar activities after ␤-glucosidase treatment. We also detected polyphenol oxidase activity in organelles that activate the dihydroxyphenolic moiety to have protein-crosslinking activities. These data suggest that the privet tree has developed an effective defense mechanism with oleuropein, a unique multivalent alkylator ideal as a protein-crosslinker. Our results that iridoid glycosides are precursors of alkylators may elucidate the chemical bases that underlie various bioactivities and ecological roles of iridoid glycosides.

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Section: Resultsmentioning

confidence: 99%

Enzymatic activation of oleuropein: A protein crosslinker used as a chemical defense in the privet tree

Konno

Hirayama²,

Yasui³

et al. 1999

Proc. Natl. Acad. Sci. U.S.A.

207

174

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“…Glutaraldehyde, which does not hydrolyze or become otherwise inactivated over long periods of time, is widely used to immobilize enzymes by cross-linking and to stabilize their adsorption to or entrapment in various materials (157,158). The nature of its reactions with proteins may involve some Schiff base formation but is clearly much more complicated than that and not completely understood (137,159,160).…”

Section: Side-chain Reactivitiesmentioning

confidence: 99%

Chemical modifications of proteins: history and applications

Means¹,

Feeney²

1990

Bioconjugate Chem.

183

170

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With roots in ancient formulations, methods for the chemical derivatization of proteins continue to expand and develop. The creation of this new journal dealing exclusively with bioconjugate chemistry was barely conceivable just a few years ago. An explosion of interest in the subject during the last decade is, however, easily seen. The tremendous growth in both the number of publications and in the number of research groups involved in these kinds of studies has been promoted by both practical interests related, for example, in some cases to possible pharmacological or medical diagnostic applications and by interest in questions of fundamental biochemical structure and function.Greatly Reagents have been designed to preserve electrostatic charge (4,5), to alter electrostatic charge (6), and to increase hydrophobicity (7,8). Reagents and procedures have been developed to decrease immunogenicity (9, 20), to increase and decrease susceptibility to proteolysis (22-23), to increase UV or visible absorbancy (14), to introduce fluorescent labels (25,16), spin labels (27), radiolabels , various metal ions (22), magnetic microspheres (22,23), and electron-dense substituents (24), to increase the content of certain low-abundance nonradioactive isotopes (25)

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“…The reaction of GA with proteins is complex (see Richards [13] and Kern [8]) and chemically well characterized derivatives have, to our knowledge, not yet been isolated. Our preliminary experiments have shown that the reagent can be used to prepare insulinperoxidase conjugates, possessing both enzymatic and immunological activity, though the latter is slight.…”

Section: Resultsmentioning

confidence: 99%

“…2) was carried out in a Boskamp Mikrophor apparatus and the enzymatic activity estimated according to Chance [8].…”

Section: Coupling Proceduresmentioning

confidence: 99%

Insulin labelled by coupling with peroxidase

1973

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Summary. By coupling purified horseradish peroxidase with glutaraldehyde and subsequent reaction with insulin, conjugates were obtained. These were partially purified by ion exchange chromatography on DEAE-cellulose. A fraction with an average molar ratio of peroxidase to insulin t :0.37 was analysed by electrophoresis and gel filtration. The peroxidase activity of this fraction was found to be 19% of normal and the immunological reactivity 0.6o as compared with that ef insulin. Peroxidase labelled insulin Can be utilized in biological studies of insulin receptors and in immunological studies of the insulin antibody-producing cells. Therefore, we coupled insulin to horseradish peroxidase {HRP) by glutaraldchyde (GA) using a slight modification of the original method described by Awame~s [3]. Coupling products with different ratio of insulin to HRP have been obtained. Materials and MethodsPeroxidase (AbR=0.651): Boehringer, Mannheim; crystallized insulin(bovine) and 125I-insulin(porcine) : Farbwerke Hoechst, Frankfurt; DEAE and CM-cellulose : Serva, Heidelberg; Sephadex G-25 and G-50: Pharmacia, Frankfurt; glutaraldehyde (GA; 25% solution): Merck, Darmstadt; bovine serum albumin: Behringwerke, Marburg. Antibodies against insulin had been prepared in our laboratories by immulaisation of guinea pigs with insulin in complete Freund's adjuvant.HRP was purified on DEAE-cellulose and subsequently on CM-celhflose according to the method described by Shannon [14]. B and C isoenzymes (AbR usually of 2.9--3.3) were pooled and a concentration reached by ultrafiltration of 50 mg HRP/ml. Crystallized insulin was purified by gel filtration on Sephadex G-50 superfine (column 65 • 1500 ram) in 10% acetic acid. 200 mg of this material was mixed with different quantities of 126I-insulin (usually 200 ~U) and applied to a DEAE-cellulose column * Kindly supported by Landesversicherungsanstalt Rheinprovinz, Diisseldorf, and Landesamt fiir Forschung, Nordrhein Westfalen.1 AbR= Absorption ratio ----r __403 nm indicating 280 nm purity grade. equilibrated with 0.005 sodium acetate buffer (pH 4.4). A linear gradient of the same buffer and 0.3 M acetate buffer, 250 ml each, was used. The insulin peak elating at a molarity of 0.2--0.3 was collected and this mixture of insulin and l~6I-insulin directly used. Coupling procedureThe method of Avrameas [3] was used with the following slight modification. A solution was prepared containing 25 mg of HRP and 12.5 mg GA per ml 0.15 M phosphate buffer (pH 6.8). After standing for 8 h at room temperature, the mixture was filtered throug h a G-25 Scphadex column (14 • 600 ram) in 0.15 M NaC1 solution. The activated HRP fraction was mixed, without any further concentration, with insulin (molar ratio 1 : 1.2) and 0.2 ml M sodium carbonate-bicarbonate buffer was added to bring the solution to pH 9.3. The resulting mixture was allowed to stand for an additional 18 h at room temperature when an excess of lysine was added to block the remaining aldehyde groups. The mixture was dialysed against 7 M urea in 0.0...

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Glutaraldehyde: Nature of the reagent

Cited by 234 publications

References 10 publications

Enzymatic activation of oleuropein: A protein crosslinker used as a chemical defense in the privet tree

Enzymatic activation of oleuropein: A protein crosslinker used as a chemical defense in the privet tree

Chemical modifications of proteins: history and applications

Insulin labelled by coupling with peroxidase

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