Affinity chromatography of thiol ester-containing proteins.
A method is described for the affinity chromatographic purification of thiol ester proteins. These comprise the complement proteins C3 and C4 and the protease inhibitor alpha 2-macroglobulin (alpha 2M) and are known to contain an internal beta-cysteinyl-gamma-glutamyl thiol ester. The method employs aminoalkyl ligands coupled to a divinylsulfonyl-derivatized agarose matrix, and the length of the aminoalkyl spacer arm was found to be important for the effectiveness of the matrix. Optimal results were obtained with diaminododecyldivinylsulfonyl-agarose. Employing this matrix the thiol ester proteins C3, C4 and alpha 2M were isolated from human pregnancy serum. Application of the method to chicken and rainbow trout serum gave rise to isolation of several proteins including chicken and rainbow trout alpha 2M.
Houen
G
G
Department of Autoimmunology, Statens Serum Institut, Copenhagen, Denmark.
Svendsen
I
I
D000818:Animals; D015153:Blotting, Western; D002846:Chromatography, Affinity / Q000379:methods*; D003176:Complement C3 / Q000737:chemistry / Q000302:isolation & purification*; D003181:Complement C4 / Q000737:chemistry / Q000302:isolation & purification*; D004591:Electrophoresis, Polyacrylamide Gel; D004952:Esters / Q000737:chemistry; D005260:Female; D006461:Hemolysis; D006801:Humans; D011247:Pregnancy; D012756:Sheep; D013438:Sulfhydryl Compounds / Q000737:chemistry; D000511:alpha-Macroglobulins / Q000737:chemistry / Q000302:isolation & purification*
D003176:Complement C3; D003181:Complement C4; D004952:Esters; D013438:Sulfhydryl Compounds; D000511:alpha-Macroglobulins