Abstract
Affinity adsorbents for bovine trypsin were prepared by covalently coupling p-(p'-amino-phenoxypropoxy)benzamidine to cellulose and to agarose. Trypsin binds to both adsorbents at pH6-8 and is released at low pH values or in the presence of n-butylamine hydrochloride. Pure beta-trypsin may be eluted from crude trypsin bound at pH8.0 to the cellulose adsorbent by stepwise elution with an acetate buffer, pH5.0. Both alpha- and beta-trypsin may be isolated by chromatography of crude trypsin on the agarose derivative in an acetate buffer, pH4.0. These two methods for purifying the trypsin are specific to the particular adsorbents. They are rapid and convenient in use. Both methods leave a mixture of the two enzymes bound to the adsorbent and release occurs only at low pH values. The effects of pH, composition and ionic strength of buffer and other variables on both purification methods are described. Affinity adsorbents of soya-bean trypsin inhibitor and of N-alpha-(N'-methyl-N'-sulphanilyl) sulphanilylagmatine bound to agarose were prepared, but were found to be of limited usefulness in the purification of trypsin.