Covalent Chromatography

achieved in the bonding step, only one of the components reacts. The other components are removed by washing and the bonded component is then released by another chemical reaction. Ideally this leaves the chromatographic material in a form that is readily regenerated. When several components react with the chromatographic material, specific isolation of individual components needs to be achieved subsequently, e.g. in the elution step (sequential elution covalent chromatography). A recent extension of covalent chromatography involves derivatization specifically of thiol-containing components by reaction with a dithiopyridyl polyethyleneglycol (PEG) reagent. This provides charge shielding effects and facilitates separation of the derivatized proteins by ion exchange chromatography.

The development of covalent chromatography is discussed below and is summarized in Table 1 in which key papers and reviews are identified. Those key papers not listed in the Further Reading section may be found in one or more of the reviews. Widespread application of the technique began after 1973, when covalent chromatography by thiol-disulfide interchange using the 2-mercaptopyridine leaving

Table 1 Milestones in the development of covalent chromatography and some key publications

1963 Fundamental paper reports the synthesis of an 'organomercurial polysaccharide'for the isolation of thiol-containing proteins and the first example of covalent chromatography (Eldjarn and Jellum).

1970 Fundamental paper reports unusual high reactivity of the thiol group of papain towards 2,2'-dipyridyl disulfide (2-Py-S-S-2-Py; 2PDS) at pH 4 which provided the basis for covalent chromatography by thiol-disulfide interchange with provision for selectivity for low pK thiol groups (Brocklehurst and Little).

1972 Fundamental paper reports an early example of covalent affinity chromatography (a combination of covalent and conventional affinity chromatographies) in which penicillin-binding proteins are isolated by reaction with the ^-lactam ring of immobilized 6-aminopenicillamic acid and released by reaction with hydroxylamine (Blumberg and Strominger).

1973 Fundamental paper introduces covalent chromatography by thiol-disulfide interchange for the isolation of fully active papain using a Sepharose-(glutathione-2-pyridyl disulfide) gel (Brocklehurst, Carlsson, Kierstan and Crook; marketed by Pharmacia).

1975 Fundamental paper reports the synthesis and use of a more highly substituted gel with an electrically neutral and less sterically demanding spacer, the Sepharose 2-hydroxypropyl-2'-pyridyl disulfide gel (Axen, Drevin and Carlsson; marketed by Pharmacia).

1978 Fundamental paper reports the introduction of A/-succinimidyl-3-(2'-pyridyl disulfanyl) propanoate which readily permits the introduction of auxiliary thiol groups into non-thiol-containing proteins to widen the scope of targets for reversible immobilization by thiol-disulfide interchange (Carlsson, Drevin and Axen).

1980 Review cites approx. 150 papers on covalent chromatography published between 1973 and 1978; although most publications are concerned with thiol-containing proteins, there are some references to covalent chromatography involving serine, methionine and tryptophan side chains and to the isolation of nucleic acids and membrane fragments (Lozinskii and Rogozhin).

1981 Fundamental paper reports development of sequential elution covalent chromatography to separate protein disulfide isomerase and glutathione insulin transhydrogenase (Hillson).

1982 Reviewdiscusses selectivity by proton-activated covalent chromatography using 2-pyridyl disulfide gels in acidic media as a logical extension of the more general use of soluble disulfides containing the 2-mercaptopyridine leaving group in protein chemistry and enzymology as enzyme active centre titrants, reactivity probes, delivery vehicles for spectroscopic reporter groups and heterobifunctional crosslinking reagents (Brocklehurst).

1983 Fundamental paper reports the use of (Gly-Phe-Phe)2-cystamine immobilized on Affi-Gel10 (BioRad) for the isolation of cathepsin B; this is an example of an extension of the general method of covalent chromatography by thiol-disulfide interchange by provision of recognition sites to create a covalent affinity gel (Evans and Shaw).

1985 Review discusses covalent chromatography and its applications in biochemistry and biotechnology; extensive detailed descriptions are given of the synthesis, characteristics and commerical sources of activated support materials (Brocklehurst, Carlsson and Kierstan).

1995 Fundamental paper reports examples of selectivity in covalent chromatography by thiol-disulfide interchange determined by steric and electrostatic restrictions (Thomas, Verma, Boyd and Brocklehurst).

1996 Reviewsummarizes applications of covalent chromatography by thiol-disulfide interchange with references also to the use of some other types of thiol-specific chromatography: organomercurials, isothiocyanates and 4-aminophenylarsenoxide-agarose for the selective isolation of molecules containing vicinal thiol groups (Brocklehurst).

1996 Fundamental paper discusses an example of a development of covalent chromatography whereby monomethoxypolyoxy-(ethylene glycol) (mPEG)-(glutaryl)-S-S-2-Py is used to derivatize components of a mixture of thiol-containing enzymes to facilitate their separation by ion exchange chromatography (Azarkan, Maes, Bouckaert, Thi, Wyns and Looze).

Table 2 Applications of covalent chromatography by thiol-disulfide interchange using 2-pyridyl disulfide-containing gels or 2-pyridyl disulfide derivatives of the target protein or peptide



Fractionation and specific isolation of thiol-containing proteins and peptides

Isolation and sequencing of thiol-containing peptides

Removal of prematurely terminated peptides during solid-phase peptide synthesis

Reversible immobilization of enzymes with associated purification

Synthesis of specific adsorbents for conventional affinity chromatography

Purification of a wide range of enzymes and other proteins by the various versions of the technique has been reported

Facilitates purification of thiol-containing peptides which is often difficult from proteolytic digests. Two versions: (i) immobilization of the protein by reaction with the disulfide gel followed by proteolysis; (ii) derivatization of the protein by reaction with 2PDS, proteolysis in solution and isolation by reaction with the thiolate gel

Premature chain termination of the peptide by blocking of the free terminal amino group results in unwanted by-products in solidphase peptide synthesis. These are readily separated from non-terminated peptides by addition of Cys-Met to the free amino group of the non-terminated peptide prior to cleavage from the solidphase matrix in preparation for covalent chromatography

This method contrasts with most methods of immobilization which are irreversible. An additional advantage is that eventual release of the enzyme by thiolysiscan produce purified enzyme if the preparation applied was not fully active

Thiol-disulfide interchange provides a convenient method of attaching ligands containing specific recognition features to insoluble matrices group was introduced by Brocklehurst et al., initially for the specific isolation of the fully active form of the cysteine proteinase, papain.

Various approaches developed subsequently are discussed including the range of gel types, the reactions involved in attachment, elution and gel reactivation and brief discussion of specific covalent attachment via groups other than thiol groups. The range of applications of covalent chromatography by thiol-disulfide interchange is summarized in Table 2.

Solar Panel Basics

Solar Panel Basics

Global warming is a huge problem which will significantly affect every country in the world. Many people all over the world are trying to do whatever they can to help combat the effects of global warming. One of the ways that people can fight global warming is to reduce their dependence on non-renewable energy sources like oil and petroleum based products.

Get My Free Ebook

Post a comment