Enzyme-linked immunosorbent assay, ELISA

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.

https://www.future-science.com/doi/pdf/10.2144/04375RV01

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

The Cross-linking of Proteins with Glutaraldehyde and its use for the preparation of immunoadsorbents

Proteins for foods, in addition to providing nutrition, should also possess specific functional properties that facilitate processing and serve as the basis of product performance. Functional properties of proteins for foods connote the physicochemical properties which govern the behavior of protein in foods. This general article collates the published information concerning the major functional properties of food proteins, e.g., solubility, binding properties, surfactant properties, viscogenic texturizing characteristics, etc. The effects of extraction and processing on functional properties and possible correlations between structure and function are discussed, in relation to practical performance in food systems. Modification of proteins to improve functional characteristics is briefly mentioned.

Functional properties of proteins in foods: A survey

Publisher Summary This chapter discusses a reaction of protein sulfhydryl groups with Ellman's reagent. The reagent 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) is developed by Ellman as a sulfhydryl reagent. DTNB is found to be a sensitive tool for the assay of thiol groups in tissues, body fluids, and proteins. Ellman described a method for its synthesis. DTNB is an aromatic disulfide, and, since it has a higher standard oxidation-reduction potential than aliphatic analogs, it will react with aliphatic thiols by an exchange reaction to form a mixed disulfide of the protein and 1 mole of 2-nitro-5-thiobenzoate per mole of protein sulfhydryl group. Sulfhydryl-disulfide exchange reactions between disulfide compounds with sulfur directly attached to aromatic groups and simple alkyl mercaptans should go to completion. Protein sulfhydryl (SH) groups may behave similarly to simple alkyl mercaptans unless steric factors interfere with the course of reaction. The sulfhydryl groups in proteins exhibit variable reactivity toward DTNB owing to steric factors. Determination of total sulfhydryl content requires that the protein be denatured, preferably with sodium dodecyl sulfate.

A.F.S.A. Habeeb

Papers

Reaction of Proteins with Glutaraldehyde

Preparation of enzymically active, water-insoluble derivatives of trypsin.

Quantitation of conformational changes on chemical modification of proteins: use of succinylated proteins as a model.

Studies on epsilon-prototoxin of Clostridium perfringens type D. I. Purification methods: evidence for multiple forms of epsilon-prototoxin.