Immobilization of aspartate aminotransferase on agarose

Biochimie
K Kurkijärvi, T Korpela

Abstract

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.

References

Jul 27, 1979·Biochemical and Biophysical Research Communications·M Arrio-Dupont, P R Coulet
May 1, 1978·Archives of Biochemistry and Biophysics·R C Harruff, W T Jenkins
Apr 1, 1977·Biotechnology and Bioengineering·L GrassetA Ville
Sep 1, 1976·Archives of Biochemistry and Biophysics·R C Harruff, W T Jenkins
Mar 1, 1976·Analytical Biochemistry·T Korpela, A Hinkkanen
Jul 1, 1974·Analytical Biochemistry·S C MarchP Cuatrecasas
May 2, 1974·European Journal of Biochemistry·J CarlssonE M Crook
Jan 1, 1980·Biochimie·P R Coulet, D C Gautheron
May 1, 1980·Analytical Biochemistry·T Korpela, K Kurkijärvi

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Citations

Nov 6, 2001·Journal of Biochemical and Biophysical Methods·V I MuronetzT Korpela
May 28, 2003·Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences·Vladimir I Muronetz, Timo Korpela
Oct 21, 1999·Methods : a Companion to Methods in Enzymology·S Beeckmans

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