Affinity labeling of a subsite of Taka-amylase A by the fluorescent reagent o-phthalaldehyde.
A cross-linked modification of Lys residue located at the subsite of the enzyme active site of Taka-amylase A was attained by the use of the fluorescent reagent of o-phthalaldehyde (OPA). The fluorescence and uv absorption at 337 nm derived from the isoindole ring, which was produced by cross-linking through the epsilon-amino group of Lys and the thiol group of the Cys residue, provided the evidence for the OPA-mediated inactivation of Taka-amylase A. Kinetic analysis showed that 1 mol of OPA per mole of enzyme was incorporated, which corresponded closely with the value obtained by the uv absorption. Because the OPA inactivation was retarded by the substrate analog of alpha-cyclodextrin, OPA modification was classified as a type of affinity labeling reaction. A remarkable increase in the pI value from 4.0 to 5.6 upon the modification led to clear separation of the modified enzyme from the native Taka-amylase A by a DEAE-Sephacel column and led to the charge isomer pattern on gel electrophoresis performed according to the method of Hedrick and Smith. Moreover, the affinity gel electrophoresis showed that the modified enzyme completely lost the affinity for the substrate soluble starch, which indicated that the subsite modification occurred.
Kobayashi
M
M
National Food Research Institute, Ibaraki, Japan.
Miura
M
M
Watanabe
T
T
Ichishima
E
E
D000345:Affinity Labels; D001236:Aspergillus oryzae / Q000201:enzymology; D001665:Binding Sites; D003432:Cross-Linking Reagents; D005456:Fluorescent Dyes; D007700:Kinetics; D008239:Lysine; D000516:alpha-Amylases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D009764:o-Phthalaldehyde / Q000494:pharmacology
D000345:Affinity Labels; D003432:Cross-Linking Reagents; D005456:Fluorescent Dyes; D009764:o-Phthalaldehyde; D000516:alpha-Amylases; D008239:Lysine