Protein purification by affinity chromatography. Derivatizations of agarose and polyacrylamide beads.

The purpose of this review is to examine the various effects of low- molecular-weight electrolytes on the associations and interactions of proteins and nucleic acids. Our primary interest is in general electrostatic effects, rather than chemical effects (specific interactions) of particular ions (e.g. transition metals, protons). We consider those interactions in which a variation in salt concentration has a significant effect on the macromolecular equilibrium, and analyse the effects of salt in these situations in terms of (i) direct participation of ions in the biopolymer reaction, (ii) Debye—Huckel screening by salt ions of the charge interactions on the biopolymers, and (iii) the reduction in water activity brought about at high salt concentrations.

https://www.cambridge.org/core/services/aop-cambridge-core/content/view/S003358350000202X

Thermodynamic analysis of ion effects on the binding and conformational equilibria of proteins and nucleic acids: the roles of ion association or release, screening, and ion effects on water activity.

Publisher Summary The selective isolation and purification of enzymes and other biologically important macromolecules by “affinity chromatography” exploits the unique biological property of the proteins or polypeptides to bind ligands specifically and reversibly Affinity chromatography exploits the phenomenon of specific biological interaction in a large variety of protein–ligand systems A solution containing the macromolecule to be purified is passed through a column containing an insoluble polymer or gel to which a specific competitive inhibitor or other ligand has been covalently attached Proteins not exhibiting appreciable affinity for the ligand pass unretarded through the column, whereas those which recognize the inhibitor are retarded in proportion to the affinity existing under the experimental conditions The specifically adsorbed protein can be eluted by altering the composition of the solvent so that dissociation occurs Affinity chromatography may be useful in concentrating dilute solutions of proteins, in removing denatured forms of a purified protein, and in the separation and resolution of protein components resulting from specific chemical modifications of purified proteins Inherent advantages of this method of purification are the rapidity and ease of a potentially single-step procedure, the rapid separation of the protein to be purified from inhibitors and destructive contaminants, such as proteases, and protection from denaturation during purification by active site ligand-stabilization of protein tertiary structure

[31] Affinity chromatography

Recombinant DNA technology has now made it possible to produce proteins for pharmaceutical applications Consequently, proteins produced via biotechnology now comprise a significant portion of the drugs currently under development Isolation, purification, formulation, and delivery of proteins represent significant challenges to pharmaceutical scientists, as proteins possess unique chemical and physical properties These properties pose difficult stability problems A summary of both chemical and physical decomposition pathways for proteins is given Chemical instability can include proteolysis, deamidation, oxidation, racemization, and β-elimination Physical instability refers to processes such as aggregation, precipitation, denaturation, and adsorption to surfaces Current methodology to stabilize proteins is presented, including additives, excipients, chemical modification, and the use of site-directed mutagenesis to produce a more stable protein species

Stability of Protein Pharmaceuticals

Many, if not most, redox reactions are coupled to proton transfers. This includes most common sources of chemical potential energy, from the bioenergetic processes that power cells to the fossil fuel combustion that powers cars. These proton-coupled electron transfer or PCET processes may involve multiple electrons and multiple protons, as in the 4 e–, 4 H+ reduction of dioxygen (O2) to water (eq 1), or can involve one electron and one proton such as the formation of tyrosyl radicals from tyrosine residues (TyrOH) in enzymatic catalytic cycles (eq 2). In addition, many multi-electron, multi-proton processes proceed in one-electron and one-proton steps. Organic reactions that proceed in one-electron steps involve radical intermediates, which play critical roles in a wide range of chemical, biological, and industrial processes. This broad and diverse class of PCET reactions are central to a great many chemical and biochemical processes, from biological catalysis and energy transduction, to bulk industrial chemical processes, to new approaches to solar energy conversion. PCET is therefore of broad and increasing interest, as illustrated by this issue and a number of other recent reviews.

/pdf/thermochemistry-of-proton-coupled-electron-transfer-reagents-v04esq6den.pdf

Reinhold Benesch

Papers

Reciprocal binding of oxygen and diphosphoglycerate by human hemoglobin

The Acid Strength of the -SH Group in Cysteine and Related Compounds

The oxygenation of hemoglobin in the presence of 2,3-diphosphoglycerate. Effect of temperature, pH, ionic strength, and hemoglobin concentration.

The preparation and properties of the isolated alpha and beta subunits of hemoglobin A.

The influence of oxygenation on the reactivity of the--SH groups of hemoglobin.