Removal of endoproteinases from biological fluids by "sandwich affinity chromatography" with alpha 2-macroglobulin bound to zinc chelate-Sepharose.
A new method for removing nearly all active endoproteinases from fluids called "sandwich affinity chromatography" is described. It is based on strong chelate binding of alpha 2-macroglobulin (alpha 2M) and its proteinase complexes to Zn2+-bis-carboxymethylamino-Sepharose (Zn chelate-Sepharose) and its ability to complex most active endoproteinases. The preferred performance minimizing unspecific protein adsorption is binding first alpha 2M to Zn chelate-Sepharose and then adsorbing the proteinase to the alpha 2M-Zn chelate-Sepharose using elevated salt concentrations. A suitable standard buffer, in which most proteases and alpha 2M are active and the protease-alpha 2M complex remains bound to Zn chelate-Sepharose, is 0.02 mol/liter sodium phosphate, pH 6.5, containing 0.15 mol/liter NaCl. As an example, the reaction of trypsin with alpha 2M-Zn chelate-Sepharose was studied. After saturating Zn chelate-Sepharose first with alpha 2M and then with trypsin under standard conditions, the bound alpha 2M equals the bound trypsin activity (measured with Chromozym TRY). The specific binding capacity of alpha 2M-Zn chelate-Sepharose for proteases was determined in this way to be 30-40 U trypsin, i.e., 0.40-0.54 mg/ml of gel. The balance and the fact that the bound trypsin is inaccessible to soybean trypsin inhibitor indicate that at these conditions no unspecific trypsin binding occurs. Chymotrypsin, thermolysin, elastase, bromelain, ficin, and papain are also bound at standard conditions but not exoproteases like carboxypeptidases A and Y. Advantages of the sandwich affinity chromatography are the simple loading procedure by adsorption, the high capacity of the gel material, and the possibility to reuse the Zn chelate-Sepharose after eluting reacted alpha 2M and reloading with new alpha 2M.
D000818:Animals; D001826:Body Fluids / Q000201:enzymology*; D002412:Cations; D002417:Cattle; D002846:Chromatography, Affinity; D010450:Endopeptidases / Q000302:isolation & purification*; D006801:Humans; D007202:Indicators and Reagents; D012685:Sepharose / Q000031:analogs & derivatives; D014357:Trypsin / Q000378:metabolism; D000511:alpha-Macroglobulins
D002412:Cations; D007202:Indicators and Reagents; D000511:alpha-Macroglobulins; C030523:zinc chelate-sepharose; D012685:Sepharose; D010450:Endopeptidases; D014357:Trypsin