The reactions between glutaraldehyde and various proteins. An investigation of their kinetics

Synopsis Glutaraldehyde reacts readily with various proteins in solution. With high concentrations of both, the solutions become yellow and many proteins form a gel. At low concentrations the reactions may be followed by the changes in the u.v. spectrum between 250 and 300 nm. The reverse reaction does not proceed to any detectable extent. The kinetics are pseudo-first-order. The activation energies for the reactions between proteins and glutaraldehyde were found to be about II kcal/mole. This suggests that the proteins have not been denatured to any marked extent by the glutaraldehyde fixation. The rates of reactions increase with pH. The rate of formation of glutaraldehyde-protein links per protein molecule glutarated is approximately I sec^–1 mol^–1 1.     

A new method for the study of glutaraldehyde-induced crosslinking properties in proteins with special reference to the reaction with amino groups.

A new method for the study of glutaraldehyde reactions with proteins is presented. Glutaraldehyde-reacted protein is in a first step isolated and then in a second step reacted with aminohexyl groups bound to Sepharose particles. This reaction is linear at low protein concentrations and proceeds rapidly when proteins are reacted with 100-fold and 1000-fold molar excess of glutaraldehyde. This method enables the study of glutaraldehyde-induced crosslinking properties of the modified proteins as an isolated property with high reliability.     

Glutaraldehyde: behavior in aqueous solution, reaction with proteins, and application to enzyme crosslinking

Glutaraldehyde possesses unique characteristics that render it one of the most effective protein crosslinking reagents. It can be present in at least 13 different forms depending on solution conditions such as pH, concentration, temperature, etc. Substantial literature is found concerning the use of glutaraldehyde for protein immobilization, yet there is no agreement about the main reactive species that participates in the crosslinking process because monomeric and polymeric forms are in equilibrium. Glutaraldehyde may react with proteins by several means such as aldol condensation or Michael-type addition, and we show here 8 different reactions for various aqueous forms of this reagent. As a result of these discrepancies and the unique characteristics of each enzyme, crosslinking procedures using glutaraldehyde are largely developed through empirical observation. The choice of the enzyme-glutaraldehyde ratio, as well as their final concentration, is critical because insolubilization of the enzyme must result in minimal distortion of its structure in order to retain catalytic activity. The purpose of this paper is to give an overview of glutaraldehyde as a crosslinking reagent by describing its structure and chemical properties in aqueous solution in an attempt to explain its high reactivity toward proteins, particularly as applied to the production of insoluble enzymes.     

Glutaraldehyde fixation chemistry: oxygen-consuming reactions

In tissue fixed with glutaraldehyde, dissolved O2 is rapidly consumed by two processes: residual respiration and glutaraldehyde-induced chemical uptake. The nature of the chemistry which consumes O2 during tissue fixation was investigated by studying model reactions of glutaraldehyde with amines and with homogenized tissue suspensions. The addition of glutaraldehyde to solutions of most primary amines and ammonia stimulated rapid O2 consumption. The reaction of glutaraldehyde with primary amines (e.g., 25 mM ethanolamine, glycine, or methylamine) consumed 50% of the dissolved O2 in 15 to 20 s at 37 degrees C. The initial rate of O2 uptake followed second-order kinetics with respect to the primary amine concentration. The total amount of O2 consumed was sufficient to account for the stoichiometric conversion of the primary amines to pyridines. These data are consistent with the synthesis of pyridine derivatives from glutaraldehyde-amine precursors in which the last step is an irreversible oxidation of dihydropyridines to pyridines. The addition of glutaraldehyde to homogenized muscle suspensions, in which respiration was chemically inhibited, significantly increased the rate of O2 uptake. Thus, in tissue O2 is rapidly depleted both by respiration and the chemical demands of the glutaraldehyde-amine reactions during the cross-linking process. Since these experiments were done under conditions commonly used for tissue fixation, hypoxia should be assumed to exist in biological preparations fixed with glutaraldehyde.     

Contributions of lipids and proteins to the surface charge of membranes. An electron microscopy study with cationized and anionized ferritin

The surface charge of cultured neurons was investigated with the electron microscope markers anionized ferritin (AF) and cationized ferritin (CF). To determine which membrane components could react with the markers, model reactions were used. Both protein-coated Sepharose beads and lipid vesicles were reacted at physiological pH. Results with these model reactions indicate that the following groups may contribute to the surface charge: acidic groups--the sialic acid of both glycoproteins and gangliosides, the carboxyl group of proteins, and the phosphates of phospholipids; basic groups--the amines of proteins. The effect of chemical fixation on the surface charge was investigated. Glutaraldehyde fixation was shown to increase the charge of neutral proteins but not by a mechanism involving unbound aldehydes. Glutaraldehyde fixation of phospholipid vesicles in the presence of CF showed that amine-containing phospholipids were cross-linked to CF. This cross-linkage was seen with the electron microscope as the clumping of CF and the burying of CF in the membrane. Paraformaldehyde fixation had a lesser effect on the charge of proteins but did react with phospholipids as did glutaraldehyde. It is concluded that at physiological pH: (a) most of the charged proteins and lipids on cell surface can contribute to the membrane surface charge, and (b) the membrane surface charge of cells can be greatly changed by chemical fixation.     

Glutaraldehyde: nature of the reagent

Aqueous solutions of glutaraldehyde used for the chemical modification and stabilization of proteins have been found to consist of free glutaraldehyde (I), the cyclic hemiacetal of its hydrate (II) and oligomers of this (III) in equilibrium with each other. Ultraviolet absorption spectra of these solutions at wavelengths greater than 200 nm should have only a single maximum at 280 nm. Absorption at 235 nm is due to an impurity which can be removed by various means. Reactions of the reagent with proteins involve principally the lysinyl (and hydroxylysinyl) residues in the relative amounts of four moles of glutaraldehyde to one of lysine. Three unstable products can be partially isolated from acid hydrolyzates of glutaraldehyde-treated proteins or from the reaction mixtures of glutaraldehyde and model compounds; two of these products have strong ultraviolet absorption near 265 nm.     

Some studies of crosslinking chitosan-glutaraldehyde interaction in a homogeneous system.

Chitosan dissolved in acetic acid reacted with glutaraldehyde solution, ranging in concentration from 0.10 to 25.0 x 10(-2) mol dm3. The modified polymers were characterized by means of carbon, hydrogen and nitrogen elemental analysis, scanning electron microscopy, X-ray diffractometry, 13C nuclear magnetic resonance (NMR), infrared and Raman spectroscopies. The uptake of metallic cations in aqueous medium was checked through copper. The obtained data from 13C NMR, infrared and Raman spectroscopies evidenced the formation of an ethylenic double bond in the chitosan glutaraldehyde interaction. These data suggest that free pendant amine groups of chitosan polymer interact with the aldehydic group of the glutaraldehyde to form stable imine bonds, due to the resonance established with adjacent double ethylenic bonds. The crosslinking is formed by the nonuniform length of chains and by terminal unities. The crosslinking formation can involve two chitosan unities belonging, or not, to the same polymeric chain. The sequence of reactions was established for a chitosan:glutaraldehyde molar proportion of 1:20. The degree of crystallinity and particle size decreased as the amount of glutaraldehyde was increased in the polymer. Physical and chemical properties are not just affected for the chitosan glutaraldehyde reaction, but are also affected strongly by the dissolution of the natural chitosan.     

Glutaraldehyde: Role in electron microscopy

In spite of the inherent limitations of chemical fixation, glutaraldehyde is unsurpassed in its ability to preserve cell ultrastructure. This achievement is due to the introduction of irreversible intra-and intermolecular cross-links into cellular proteins by the dialdehyde. Glutaraldehyde is very effective in stabilizing surface as well as intracellular structures for conventional scanning and transmission electron microscopy and high voltage electron microscopy. Even in immunocytochemical and autoradiographical studies, glutaradehyde plays a dominant role. Furthermore, prior to freeze-substitution, freeze-drying and freeze-fracturing, specimens often are stabilized with this dialdehyde. Glutaraldehyde efficiency can be increased by adding appropriate cross-linking and rapidly penetrating reagents as well as contrast enhancing reagents to this dialdehyde. Improved preservation and staining, for example, of ionic sites, soluble inorganic phosphate, lipids, biogenic amines, actin filaments, spermatozoa and phage-infected bacteria can be accomplished by adding polyethyleneimine, lead acetate, malachite green, potassium dichromate, tannic acid, trinitro compounds and uranyl acetate, respectively, to glutaraldehyde. Other refinements include the use of low concentrations of glutaraldehyde, short durations of cross-linking, minimum radiation exposure, and low temperature electron microscopy. The usefulness of glutaraldehyde in high resolution electron microscopy is limited because chemical fixation inevitably causes chemical and structural alterations in the specimen. However, fixation with glutaraldehyde or its mixture with formaldehyde has served immeasurably the progress in the understanding of cell ultrastructure and function. Preservation of specimens with glutaraldehyde for electron microscopy is expected to continue. Therefore, attempts must continue to be made to interpret the dynamics of the living cell from the static electron micrographs.     

Photosynthetic Light Reactions in Chemically Fixed Anacystis nidulans, Chlorella pyrenoidosa, and Porphyridium cruentum

The photochemical activities of various species of unicellular algae (Anacystis nidulans, Chlorella pyrenoidosa, and Porphyridium cruentum) were studied following chemical fixation. Fixation with formaldehyde and glutaraldehyde yielded cells which retained their ability to perform photosystem I and photosystem II reactions. The photochemical efficiencies of some fixed algae are as great as those of unfixed spinach chloroplasts. Fixed algae containing accessory pigments appear to be useful models for further studies of the light reactions of photosynthesis.     

Étude du mécanisme d'établissement des liaisons glutaraldéhyde-protéines

Commercial aqueous 25 per cent glutaraldehyde solutions contain no stable derivative of this aldehyde, but compounds of variable molecular weight which easily revert to glutaraldehyde. The effect of pH on the reaction of glutaraldehyde with amino acids and on the stability of the products under acid conditions, shows the importance of the structure modification of the dialdehyde which occurs when pH increases, and even leads to precipitation in highly alkaline solutions. This precipitate results from aldol condensation of glutaraldehyde molecules. It contains aldehyd groups conjugated with ethylenic double bonds. Such a structure reacts with amino groups to give an imino bond, stabilized by resonance with the ethylenic bond, and does not undergo Michael-type addition reactions.Therefore, glutaraldehyde does not react with proteins under its free form, but as an unsaturated polymer, which gives imino bonds stabilized by conjugation.     

Biochemical aspects of aldehyde fixation and a new formaldehyde fixative.

In this paper, it is assumed that tissue fixation is a process in which the proteins become less soluble and catabolic reactions stop. With this definition in mind, 2.5 and 5% glutaraldehhde and 4% formaldehyde in 0.1 M potassium phosphate buffer, pH 7.4, were compared with a new fixative, bicarbonate-formaldehyde. The following results were obtained. (I) With 2.5 and 5% glutaraldehyde, the solubility of tissue proteins were not decreased unifromly, and tissue glycogen was poorly preserved. (2) 4% formaldehyde in potassium phosphate buffer gave relatively good results. (3) Bicarbonate-formaldehyde decreased the solubility of tissue proteins reliably and preserved tissue glycogen perfectly. Histologically, it yielded excellent results. Since glutaraldehyde alters the properties of proteins substantially (Hopwood, 1972; Habeeb & Hiramoto, 1968), and since the natural appearance of tissues depends on native tissue proteins, formaldehyde-containing fixatives, in particular bicarbonate-formaldehyde, are preferable to glutaraldehyde-containing fixatives for all tissue preparative techniques. However, it is important that the fixation time in formaldehyde is kept short.     

Influence of fixation procedures on the microanalysis of lead-induced intranuclear inclusions in rat kidney

Using Laser Microprobe Mass Analysis (LAMMA), we studied the chemical composition of lead-induced intranuclear inclusions in rat kidney tissue prepared by three different wet chemical fixation procedures for transmission electron microscopy. Fixation with glutaraldehyde-Na2S gave the same results as fixation with glutaraldehyde only: a high lead concentration could be detected. Therefore, for lead strongly bound to proteins, precipitation procedures are not essential. Post-fixation with osmium tetroxide drastically changed the composition of the inclusions: the lead concentration decreased substantially, while sodium, calcium, and barium were introduced. The osmium tetroxide fixative was found to be the source of the contamination. It also contained aluminum, and we suggest that other proteins (e.g., in neurofibrillary tangles) might be able to take up Al out of solution and that care must be exercised in interpreting the microanalytical results of osmium-fixed material. For the microanalysis of the lead inclusions, fixation with glutaraldehyde only provides a good compromise between preservation of the ultrastructure and maintenance of the element distribution.     

Immobilization of glucoamylase on polyamide nets

The formation of reactive groups on polyamide nets (nylon 6) and the subsequent immobilization of glucoamylase were investigated. Different mesh sizes of the nets and two chemical methods of enzyme coupling - i( partial hydrolysis of the polyamide with subsequent glutaraldehyde binding and ii) O-alkylation of the carrier using a treatment with a benzene-methyl sulphate mixture – were used. The reactivity of immobilized glucoamylase (GA) was tested by hydrolysis reactions using 1% starch solutions. The highest reactivity (140 μg glc/)min × cm² was obtained for methylated nylon samples attached to a glass rod and by coupling glucoamylase on the nylon surface which had been treated with lysine and glutaraldehyde. This method resulted in a more reactive and more stable preparation of immobilized glucoamylase as compared to a simpler method of coupling glutaraldehyde to partially hydrolyzed nylon.     

The Fibrinogen Concentration in Blood of Dairy Cows and its Influence on the Interpretation of the Glutaraldehyde and Formol-Gel Test Reactions

It has earlier been shown that the formol-gel test on serum and glutaraldehyde test on whole blood are simple and rapid methods for evaluation or the immunoglobulin status in the cow. Both tests function as coagulation tests in which aldehyde groups oross-link basic blood globulins at their NH2-groups, forming polymerisates. The glutaraldehyde has in whole blood the capacity to polymerize not only immunoglobulins but also fibrinogen. This investigation was made in order to study whether the fibrinogen level may influence the result of the glutaraldehyde test, so revealing any differences between the results of that and the formol-gel test carried out on serum. In 92 cows with a variety of clinical disorders (most of them with inflammatory processes) the total protein, albumin, total globulin concentration and albumin/globulin ratio in serum and fibrinogen concentration in plasma were recorded. The material was grouped according to glutaraldehyde and formol-gel test reactions. It is shown that increases in the fibrinogen level have an effect on the results of the glutaraldehyde test. A positive glutaraldehyde test in more acute processes is ascribed to a heavy rise of plasma fibrinogen in its capacity of acute-phase protein. A positive glutaraldehyde test in chronic diseases may be viewed as a result of interaction between high immunoglobulin concentrations and elevated fibrinogen concentration. In conclusion the fibrinogen and immunoglobulin status of blood is important to assess in many diseases of cattle. The semiquantitative tests described for field use can separately, or especially in parallel use, provide valuable information about the character and development of a disease and may be regarded as good substitutes for the sedimentation rate (SR), which is not demonstrable in cattle. kw|Keywords|k]bovine fibrinogen; k]bovine serum proteins; k]formol-gel reaction; k]glutaraldehyde test; k]acute and chronic inflammations     

A study of staining for electron microscopy using collagen as a model system—VII. The effect of formaldehyde fixation

Exposure to formaldehyde brings about small but readily detectable changes in the staining behaviour of collagen fibrils. These changes can be interpreted in chemical terms by comparing fibril staining patterns with artificial patterns computer-generated from sequence data. Positive staining with phosphotung-state (where heavy metal is confined to anions), shows that most of the lysyl and hydroxylysyl side-chains lose their charge character as a result of formaldehyde treatment and cease to take up staining ions. The charge character of arginyl (and probably histidyl) residues is unaltered and these residues continue to react with stain. Acidic residues are also unaffected. These results accord with biochemical evidence that the initial reaction between proteins and formaldehyde leading to subsequent cross-linking involves modification of ε-amino (and α-amino) groups. They show too that the secondary condensation producing the actual cross-link does not alter the charge character of the second group, at least when it is on an arginyl (or histidyl) side-chain.Formaldehyde-induced changes in stain deposition can also be detected after negative staining, although they are slight compared with those brought about by glutaraldehyde. Unlike glutaraldehyde, formaldehyde introduces no bulky polymeric adducts into the fibril structure, and the conspicuous stain-excluding bands seen in negative staining patterns following glutaraldehyde fixation are absent after exposure to formaldehyde. For this reason, where chemical fixation is used to stabilize macromolecules and supramolecular aggregates prior to negative staining and high resolution electron optical imaging, formaldehyde would seem to be preferable to glutaraldehyde. Data from fibril staining patterns and from thermal stability measurements (made on collagen gels) show that formaldehyde fixation does not preclude a subsequent reaction with glutaraldehyde.As with other fixatives, there is reduced accessibility to stain after formaldehyde treatment. Accessibility is least in the overlap zone where the denser packing of collagen molecules provides greater opportunities for intermolecular cross-linking. Gel electrophoresis confirms that formaldehyde-induced cross-links in fibrils are predominantly intermolecular.     

The impact of different fixation procedures on staining of macromolecules in a microtiter system

Summary The effects of formaldehyde, glutaraldehyde, methanol, Clarke's fixative and microwave irradiation on the quantitative staining of proteins (Naphthol Yellow S) and nucleic acids (Ethyl Green—Pyronin) in a cell culture system have been investigated. Overall, glutaraldehyde rapidly yielded the highest and most consistent levels of staining when compared to all other chemical fixatives. Although microwave irradiation was found to be uneven, 4 min exposure to 700W was found to give higher levels of protein staining than those achieveable with glutaraldehyde. Time-dependent processes were observed with all procedures. In addition, dissociations in the trends of protein and nucleic acid staining were observed. It is suggested that these results domonstrate fixation events that have not previously been resolved from the effects of reagent penetration into tissue blocks.     

The interaction of glutaraldehyde with poly(α,L-lysine), n-butylamine,and collagen. II. Hydrodynamic,electron microscopic,and optical investigations on the reaction products

Interactions of glutaraldehyde with either n-butylamine, poly(α,L -lysine), or collagen resulted in a fast release of protons in dilute aqueous solutions at various pH values, followed by much slower changes. The latter reactions, which extended over hours and days, were followed spectrophotometrically and revealed the formation of distinct absorption bands in the visible and near-ultraviolet regions in all the above systems. The visible-range bands disappeared upon treatment with sodium borohydride. A qualitative relationship between oxygen uptake by the system n-butylamine–glutaraldehyde and the slow formation of colored products has been established, while the chemical nature of the reaction products has not been determined. Sedimentation velocity, viscosity, and optical rotation measurements on the products of interaction between poly(L -lysine) and glutaraldehyde in aqueous solution indicated large conformational changes in the polyamino acid present in excess (in residues) over the dialdehyde. In particular, the intrinsic viscosity dropped considerably after interaction, indicating intramolecular crosslinking. At molar ratios of 1:1 between polylsine residues and aldehyde groups, intermolecular crosslinking of polylysine was obtained at pH 8.6. Electron microscopic examinations of collagen samples treated by glutaraldehyde at various pH values indicated changes from unordered to more ordered structures upon treatment with glutaraldehyde, in particular at pH 10. The present structural and optical investigations are considered to be relevant to tanning processes of hides and to fixation procedures.     

The reactions between formaldehyde,glutaraldehyde and osmium tetroxide,and their fixation effects on bovine serum albumin and on tissue blocks

Summary The reactions between osmium tetroxide and glutaraldehyde and formaldehyde were investigated. It was found that they react together to form intermediate products which then break down to form osmium black. Glutaraldehyde reacts much more rapidly with osmium tetroxide than formaldehyde. The rates of the reactions are increased by increasing the glutaraldehyde concentration or adding bovine serum albumin to the reaction mixture. The reaction rates increase with temperature. The mixtures of fixatives were also tried on tissues and the results paralleled the model experiments. The crosslinking of bovine serum albumin by osmium tetroxide, formaldehyde and glutaraldehyde singly and in mixtures was quantitatively assessed by viscosimetry, gel filtration and disc electrophoresis coupled with densitometry. The crosslinking of bovine serum albumin by pairs of fixatives was less than that produced by the most effective of the pair. After 5 min reaction osmium tetroxide was the most effective crosslinking agent according to viscosimetric experiments, but after one hour's reaction with bovine serum albumin, glutaraldehyde was revealed as the most effective crosslinking agent by gel filtration and electrophoresis.     

Genome scale enzyme-metabolite and drug-target interaction predictions using the signature molecular descriptor

MOTIVATION: Identifying protein enzymatic or pharmacological activities are important areas of research in biology and chemistry. Biological and chemical databases are increasingly being populated with linkages between protein sequences and chemical structures. There is now sufficient information to apply machine-learning techniques to predict interactions between chemicals and proteins at a genome scale. Current machine-learning techniques use as input either protein sequences and structures or chemical information. We propose here a method to infer protein-chemical interactions using heterogeneous input consisting of both protein sequence and chemical information. RESULTS: Our method relies on expressing proteins and chemicals with a common cheminformatics representation. We demonstrate our approach by predicting whether proteins can catalyze reactions not present in training sets. We also predict whether a given drug can bind a target, in the absence of prior binding information for that drug and target. Such predictions cannot be made with current machine-learning techniques requiring binding information for individual reactions or individual targets.     

Cryosectioning and immunolabeling

In this protocol, we describe cryoimmunolabeling methods for the subcellular localization of proteins and certain lipids. The methods start with chemical fixation of cells and tissue in formaldehyde (FA) and/or glutaraldehyde (GA), sometimes supplemented with acrolein. Cell and tissue blocks are then immersed in 2.3 M sucrose before freezing in liquid nitrogen. Thin cryosections, cut in an ultracryotome, can be single- or multiple immunolabeled with differently sized gold particles, contrasted and viewed in an electron microscope. Semi-thin cryosections can be used for immunofluorescence microscopy. We describe the detailed procedures that have been developed and tested in practice in our laboratory during the past decades.