title: Purification and covalent coupling of calf brain prolidase.
abstract: We have investigated methods of stabilizing prolidase by chemical modification and covalent coupling to various supports, for use in protein hydrolysis and possible use in enzyme replacement therapy. Purified acetone powder of calf brain prolidase was further purified by gel filtration on Sephadex G-200 and chromatography on DEAE-Sephadex A25. Polyacrylamide gel electrophoresis showed that the number of bands was reduced from 11 to 2. Since yields were low, the purified (NH4)2SO4 fraction was used in all experiments. Thiolation of the enzyme reduced the amount of protein coupled to AH- or CH-Sepharose 4B. Activities were highest when the protein was linked through its carboxyl groups. The coupled enzyme showed much greater thermal stability than its free counterpart. Of the bound preparations, the thiolated was less stable than the untreated. Untreated and thiolated enzymes bound to either matrix showed higher activity at low pH and less at high pH than the free material. Thiolation shifted the pH maximum from 6.8 to 7.5. The free thiolated enzyme and that bound to activated SH-Sepharose 4B showed greater thermal stability and a broader pH range of optimal activity than the bound untreated enzyme. These results show that prolidase can be immobilized by coupling to an insoluble matrix through various types of covalent bonds with retention of activity and increased stability.
mesh_terms: D000818:Animals; D001921:Brain / Q000201:enzymology*; D002417:Cattle; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004150:Dipeptidases / Q000302:isolation & purification*; D004800:Enzymes, Immobilized / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D011392:Proline / Q000302:isolation & purification; D012685:Sepharose; D013696:Temperature; D013876:Thiophenes
chemical_list: D004800:Enzymes, Immobilized; D013876:Thiophenes; C000486:citiolone; D012685:Sepharose; D011392:Proline; D004150:Dipeptidases; C022449:proline dipeptidase