Other Types of Covalent Chromatography

Attachment via thiol groups Substitution of the 2-mercaptopyridine leaving group by other aromatic mercapto groups results in the loss of selectivity at low pH and does not appear to offer substantive advantage. The intramolecular agarose thiolsulfinates introduced by Carlsson and his colleagues in the mid-1990s provide an alternative to the mixed agarose-aromatic disulfide gels discussed above. Thiolated agarose is subjected to mild oxidation by potassium ferricyanide to produce disulfide groups followed by further oxidation to thiolsulfinate groups by a stoichiometric amount of magnesium mono-peroxyphthalate. These gels also lack the opportunity to provide selectivity for low pKa thiol groups at low pH. They do not require external leaving groups but because of that do not offer the possibility of measurement of reactive site content by thiolysis and spectral analysis. An example of covalent affinity chromatography using a substrate-like symmetrical disulfide (Gly-Phe-Phe-)2-cystamine immobilized by amide bond formation on AfR-Gel 10 was reported by Evans and Shaw in 1983. In this type of approach specific binding interactions align the nucleophilic (thiolate) and electrophilic (disulfide) reactants for the covalent bonding process.

In some applications thiol groups exist as part of the support material and one example involving reaction of thiol-agarose gels with thiol-proteins de-rivatized as mixed disulfides by reaction with 2PDS (Figure 2E) constitutes one of the alternative versions of covalent chromatography by thiol-disulfide interchange. A different application of thiol-agarose gels is in studies on nucleic acids. Cytosine and uracil residues in polynucleotides can be mercurated without appreciable change in function. These derivatives form mercaptides by reaction with thiol-agarose and are eluted subsequently by treatment with a low molecular weight mercaptan.

Attachment by reaction of thiol groups is not restricted to reaction at electrophilic sulfur. The higher reactivity of arylisothiocyanates towards thiol groups than towards amines permits their use in thiol-selec-tive covalent chromatography. An immobilized ter-valent organoarsenical, 4-aminophenylarsenoxide-agarose, has been used for the selective isolation of molecules and assemblies containing vicinal thiol groups (lipoic acid and the 2-oxoglutarate dehydro-genase multienzyme complex of which lipoic acid is a covalently bonded co-factor). Attachment involves cyclic dithioarsenite formation. Elution by 2,3-dimer-captopropane-1-sulfonic acid releases the reduced (dimercaptan) form of lipoic acid.

Attachment via seryl hydroxy groups Organophos-phate agarose derivatives are an obvious choice for isolation of proteins with highly reactive seryl hy-droxy groups such as the serine hydrolases. Coupling of 2-aminoethyl 4-nitrophenyl methyl phosphonate to succinylated aminoagarose produced a material that reacted specifically with serine hydrolases such as acetylcholine esterase and chymotrypsin. The problem with these gels is the very slow release of the enzymes even by good nucleophiles that provide reactivation in analogous soluble systems.

Attachment via methionyl thioether groups The known selectivity of alkylating agents for methionyl residues in acidic media to produce sulfonium derivatives and the possibility of regeneration by sulfur nucleophiles led Schechter et al. in 1977 to produce a chloroacetamidoethyl polyacrylamide derivative for the isolation of proteins via methionyl side chains. The relatively severe conditions required for attachment (low pH and long reaction times) limit the applications of this method. The methionyl residue cannot be at the C-terminus because such residues are converted to homoserine residues and attachment is not achieved. Regeneration of the covalent chromatography material is not provided for in this method.

Attachment via tryptophanyl side chains Arylsul-fenyl chlorides and sulfur monochloride (S2Cl2) selectively modify tryptophan residues in acidic media to form 2-arylsulfenyl tryptophan and 2-mercaptotryp-tophan moieties respectively. Rubinstein et al. used this knowledge in 1976 to prepare polyacrylamide derivatives that react covalently with tryptophan-containing peptides which are released in modified forms by treatment with a low molecular weight mercaptan. The tryptophan side chain is converted to a 2-mercaptotryptophan side chain in the process. The method could find application in protein sequencing but is of limited use for protein isolation not only because of the necessary introduction of the mercapto group but also because of the requirement for acid stability of the protein.

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