Immobilization of aspartate aminotransferase on agarose.
Various methods for immobilization of aspartate aminotransferase (AspAT; from cytosolic fraction of pig heart) on agarose were tested. Aldehyde-, thiol-, and CNBr-activated agaroses were studied in detail. The capacity of the aldehyde support to firmly bind protein was less than 0.2 mg/ml, whereas the apparent remaining specific activity of the bound AspAT was high (50-63% of soluble AspAT). The maximum capacity of SH-agarose to bind enzymatic protein was 3 mg/ml; the apparent remaining activity was 30-40%, and the specific activity determined by Vmax was 51%. Chemical coupling on to thiol-agarose did not denature the enzyme, as 93% of protein and 83% of the activity were recovered after release of the enzyme from the support. Enzyme protein was quantitatively bound to CNBr-activated agarose (up to 10 mg/ml of the gel). The apparent specific activities were 27-35%, while the value calculated from Vmax was 46%. Active site-protecting agents within the CNBr-coupling were tested. Bromphenol blue increased the apparent specific activity to 60% and Vmax to 80% at 3-fold molar concentration at the active sites. Kinetic constants for immobilized preparations were determined.
Kurkijärvi
K
K
Department of Biochemistry, University of Turku, Finland.
Korpela
T
T
D000447:Aldehydes; D000818:Animals; D001219:Aspartate Aminotransferases / Q000378:metabolism*; D001665:Binding Sites; D003488:Cyanogen Bromide; D003600:Cytosol / Q000201:enzymology; D004800:Enzymes, Immobilized / Q000378:metabolism*; D007700:Kinetics; D009206:Myocardium / Q000201:enzymology*; D012685:Sepharose; D013438:Sulfhydryl Compounds; D013552:Swine
D000447:Aldehydes; D004800:Enzymes, Immobilized; D013438:Sulfhydryl Compounds; D012685:Sepharose; D001219:Aspartate Aminotransferases; C018475:butyraldehyde; D003488:Cyanogen Bromide