Purification of several proteolytic enzymes by tosyl- and carbobenzoxy-triethylene-tetramine-sepharoses.
Tosyl-triethylenetetramine-Sepharose (Tos-T-Sepharose) and carbenzoxytriethylenetetramine-Sepharose (Z-T-Sepharose) were found to be adsorbents utilizable in the purification of several microbial and animal proteases. The former Sepharose derivative adsorbed alpha-chymotrypsin, trypsin, subtilisin, thermolysin and neutral subtilopeptidase at neutral pH range, and acid proteases such as pepsin and Rhizopus niveus protease at pH 3.5-6.5. alpha-Chymotrypsin and trypsin were eluted with 0.1 N acetic acid and Rhizopus protease with 0.5 N acetic acid, thermolysin with 1 M guanidine-HCl or 33% ethyleneglycol, whilst pepsin was recovered by elution with 2 M guanidine-HCl at pH 3.5. The binding of neutral subtilopeptidase and subtilisin to this adsorbent was comparatively weak and both the enzymes were recovered by elution with 0.5 M NaCl at neutral pH. On the other hand, Z-T-Sepharose was found to bind tightly to these proteolytic enzymes except neutral subtilopeptidase. Trypsin and alpha-chymotrypsin were released from the adsorbent column with 1 M p-toluenesulfonate, and subtilisin with 1 M guanidine-HCl or 33% ethyleneglycol at neutral pH region. By these chromatographic procedures, the specific activities of these proteolytic enzymes increased effectively. Comparison of the binding abilities of acetyl-, benzoyl-, tosyl- and carbobenzoxy-T-Sepharoses to these enzymes suggests that hydrophobicity of tosyl and carbobenzoxy groups plays an important role in the enzyme-adsorbent interaction.
D000107:Acetylation; D000818:Animals; D002845:Chromatography; D002918:Chymotrypsin / Q000302:isolation & purification; D006863:Hydrogen-Ion Concentration; D010434:Pepsin A / Q000302:isolation & purification; D010447:Peptide Hydrolases / Q000302:isolation & purification*; D011134:Polysaccharides; D012685:Sepharose / Q000031:analogs & derivatives / Q000138:chemical synthesis; D013381:Subtilisins / Q000302:isolation & purification; D013820:Thermolysin / Q000302:isolation & purification; D014357:Trypsin / Q000302:isolation & purification
D011134:Polysaccharides; D012685:Sepharose; D010447:Peptide Hydrolases; D013381:Subtilisins; D002918:Chymotrypsin; D014357:Trypsin; D010434:Pepsin A; D013820:Thermolysin