Affinity chromatography of alpha-chymotrypsin, subtilism, and metalloendopeptidases on carbobenzoxy-L-phenylalanyl-triehtylenetetraminyl-sepharose.
Carbobenzoxy-L-phenylalanyl-triethylenetetraminyl-Sepharose (Z-L-Phe-T-Sepharose) was found to be an effective affinity adsorbent for bovine pancreatic alpha-chymotrypsin [EC 3.4.21.1] as well as neutral [EC 3.4.24.4] and alkaline [EC 3.4.21.14] proteases of Bacillus species. These enzymes were adsorbed in the neutral pH range. alpha-Chymotrypsin was recovered by elution with 0.1 A acetic acid while neutral subtilopeptidase was eluted with 0.5 M NaCl at pH 0. Thermolysin and subtilisin were found in eluates with 1.5 and 2.0 M guanidine-HCl at pH 7.2, respectively. The resulting enzymes appeared homogeneous on disc-electrophoresis and showed higher specific activities than those of crystalline or highly purified preparations available commercially. Modifications of the active site serines of alpha-chymotrypsin and subtilisin by treatment with diisopropylfluorophosphate (DFP) or phenylmethanesulfonyl fluoride (PMSF) resulted in loss in their binding abilities to the adsorbent. Complexes of porcine alpha2-macroglobulin with each of these four enzymes and that of Streptomyces-subtilisin inhibitor (S-SI) with subtilisin were also found in nonadsorbed fractions.
D000818:Animals; D001407:Bacillus / Q000201:enzymology; D001665:Binding Sites; D002417:Cattle; D002846:Chromatography, Affinity / Q000295:instrumentation*; D002918:Chymotrypsin / Q000032:analysis*; D004589:Electrophoresis, Disc; D005029:Ethylenediamines; D006863:Hydrogen-Ion Concentration; D007531:Isoflurophate; D008259:Macroglobulins; D010179:Pancreas / Q000201:enzymology; D011134:Polysaccharides / Q000031:analogs & derivatives*; D012685:Sepharose / Q000031:analogs & derivatives*; D012965:Sodium Chloride; D013381:Subtilisins / Q000032:analysis*; D013552:Swine; D013820:Thermolysin / Q000032:analysis*
D005029:Ethylenediamines; D008259:Macroglobulins; D011134:Polysaccharides; D007531:Isoflurophate; D012965:Sodium Chloride; D012685:Sepharose; D013381:Subtilisins; D002918:Chymotrypsin; D013820:Thermolysin