Purification of alpha1-antitrypsin from plasma through thiol-disulfide interchange.
1. Monomeric nu-chains were conjugated with CNBr-activated Sepharose 4B. The C-terminal cysteine of the conjugated nu-chain was converted to a mixed disulfide with 3-carboxy-4-nitro-benzenethiol (Nbs) and used to separate plasma proteins with reactive thiol groups. The plasma proteins, alpha1-antitrypsin and prealbumin have the greatest affinity for the interchange reaction with mixed disulfides. The disulfide link between alpha1-antitrypsin and nu-chain is sensitive to excess Nbs, and is selectively cleaved in the presence of 5,5'-dithiobis(2-nitrobenzoate) (Nbs2) which accepts the sulfhydryl group of alpha1-antitrypsin. 2. A Simple method developed for the isolation of human alpha1-antitrypsin was equally effective for the various inherited phenotypes and for alpha1-antitrypsin from the dog, baboon, and monkey, Glutathione-Sepharose was also used successfully, but the nu-chain conjugate yielded alpha1-antitrypsin less contaminated with mercaptalbumin and prealbumin. 3. The alpha1-antitrypsin is harvested from this procedure as a mixed disulfide with Nbs. The negative charge of Nbs at pH 8.1 causes an increased electrophoretic mobility of the alpha1-antitrypsin derivative. Mild reduction liberates Nbs and electrophoretic mobility of alpha1-antitrypsin returns to normal. The method described can increase the alpha1-antitrypsin content of a plasma fraction from 5% of the total protein to 95% within one day with a yield of about 50%. This purification procedure does not exert any detectable effect on microheterogeneity.
D000818:Animals; D001665:Binding Sites; D002846:Chromatography, Affinity; D004220:Disulfides; D004285:Dogs; D000882:Haplorhini; D006801:Humans; D046911:Macromolecular Substances; D010215:Papio; D011485:Protein Binding; D013053:Spectrophotometry; D013056:Spectrophotometry, Ultraviolet; D013438:Sulfhydryl Compounds; D000515:alpha 1-Antitrypsin / Q000302:isolation & purification*
D004220:Disulfides; D046911:Macromolecular Substances; D013438:Sulfhydryl Compounds; D000515:alpha 1-Antitrypsin