Thiol-disulfide interchange chromatography using sepharose-linked thiol compounds to separate plasma proteins

Abstract

The terminal cysteinyl of light Ig chains of the κ type (κ-chains) can be used for separation of plasma protein by thiol-disulfide interchange reactions. The interaction between Sepharose-conjugated κ-chains and α 1-anti-trypsin ( α 1AT) was studied with the cysteine of one or both of the two proteins either reduced or activated by 5,5′-dithiobis-(2-nitrobenzoic acid) (Nbs 2). The largest amounts of complexes were formed with the terminal κ-chain cysteine activated by Nbs 2 and with the single cysteine of α 1-anti-trypsin reduced. Cysteine and five related thiol compounds were conjugated with Sepharose 4B or 6B to explore the preparative use of this type of interchange reaction. The capacity of the Nbs 2-activated form of κ-chains and of the six thiol compounds to link plasma proteins was studied. Bound proteins were eluted after reduction with 2-mereaptoethanol and were analyzed immunochemically for 10 of the plasma proteins having more or less reactive thiols. The elution patterns varied widely. 3-Thio-2-hydroxypropyl-Sepharose showed very high selectivity for linkage of albumin at pH 8.1, while cysteine preferably linked α 1AT and IgA. Substitution of the carboxylate group of cysteine increased the linkage of albumin. Thus, Sepharose-linked thiol compounds seem useful for the fractionation of proteins with reactive thiols and for the separation of proteins with and without reactive thiols.