The effectiveness of a new immobilized cationic triazine dye was investigated alongside two new amphoteric triazine dyes and two well known anionic triazine dyes, Procion Red H-3B and Procion Blue H-B, as chromatographic media for binding four familiar proteases-trypsin, chymotrypsin, thrombin and carboxypeptidase-B-as well as a typical oxidoreductase, lactate dehydrogenase, and human serum albumin. The new affinity adsorbent, CL-Sepharose-immobilized Cationic Dye, specifically binds trypsin-like proteases such as trypsin, thrombin, and carboxypeptidase-B, but none of the other proteins tested. In contrast, the amphoteric and anionic immobilized dyes bind all the other proteins tested in a similar fashion. The specificity of the cationic dye was exploited in the resolution of trypsin and chymotrypsin from a crude activated bovine pancreatic extract. The procedure described here affords trypsin with specific activity of 7400 units/mg with a 79% overall yield in a single step. The immobilized cationic dye, unlike previously reported adsorbents for trypsin, is inexpensive, readily synthesized, and displays a workable capacity of 4000 trypsin units or 0.55 mg protein/g moist weight gel (1.2 mumol dye/g moist weight gel) from a crude bovine pancreatic extract and, thus, is potentially amenable to process-scale operations.
