title: Hydrophobic-ionic chromatography. Its application to purification of porcine pancreas enzymes.
abstract: 1. All the porcine pancreas enzymes tested, regardless of their pI's were adsorbed on Amberlite CG-50 (a weakly acidic cation exchange resin) at pH 4, where the ion-exchange group (carboxyl group) is not dissociated. The adsorption is hardly influenced by ionic strength. 2. At pH 4, the adsorbed enzymes were partially eluted by organic solvents such as 50% propanol. 3. The adsorbed enzymes were effectively eluted by increasing the pH from 4 to 6. Trypsin (pI 10.5) was eluted before carboxypeptidase A (pI 4.5 AND 5.3) WITH 0.5 M acetate buffer, whereas the former enzyme was eluted after the latter enzyme with 0.2 M 3,3-dimethyl glutarate buffer. However, with either buffer, the elution order of enzymes was not always the same as the order of the pI's. 4. By a single Amberlite CG-50 column chromatography of porcine pancreas extracts, kallikrein, carboxypeptidase B, deoxyribonuclease, carboxypeptidase A, and trypsin were purified 100-fold, 16-fmately 13%. The purification procedures included treatment with protamine, ammonium sulfate fractionation, treatment with acid, DE-32 cellulose column chromatography, gel filtration on Sephadex G-100, preparative polyacrylamide gel electrophoresis, and affinity chromatography on 5' AMP-Sepharose 4B. The last procedure, affinity chromatography on 5' AMP-Sepharose 4B, was useful for the removal of other dehydrogenases. The enzyme which was homogeneous, as shown by polyacrylamide gel electrophoresis, had a molecular weight of about 92,000. The optimum pH was at 10.0 and isoelectric point at 5.2. The enzyme accepted both L-fucose and D-arabinose as substrate, but was specific for NAD+ as coenzyme. Km values were 0.15 mM, 1.4 mM, and 0.07 mM for L-fucose, D-arabinose, and NAD+, respectively. A single enzyme catalyzed the oxidation of L-fucose and D-arabinose, which had the same configurations of hydroxyl groups from C-2 to C-4. The reaction products obtained with L-fucose as substrate were L-fucono-lactone and L-fuconic acid. The L-fucono-lactone was an immediate product of oxidation and was hydrolyzed to L-fuconic acid spontaneously. This reaction was irreversible. Therefore, it is likely that L-fucose dehydrogenase is involved in the initial step of the catabolic pathway of L-fucose in rabbit liver.
mesh_terms: D000681:Amylases / Q000302:isolation & purification*; D000818:Animals; D002268:Carboxypeptidases / Q000302:isolation & purification*; D002852:Chromatography, Ion Exchange / Q000379:methods; D003851:Deoxyribonucleases / Q000302:isolation & purification*; D007610:Kallikreins / Q000302:isolation & purification*; D008049:Lipase / Q000302:isolation & purification*; D010179:Pancreas / Q000201:enzymology*; D012997:Solvents; D013552:Swine; D014357:Trypsin / Q000302:isolation & purification*; D000516:alpha-Amylases / Q000302:isolation & purification*
chemical_list: D012997:Solvents; D003851:Deoxyribonucleases; D008049:Lipase; D000681:Amylases; D000516:alpha-Amylases; D002268:Carboxypeptidases; D007610:Kallikreins; D014357:Trypsin