Continuous spectrophotometric assays for beta-glucosidases acting on the plant glucosides L-picein and prunasin.
The neutral pH optimum beta-glucosidases of mammalian liver and almonds are each capable of hydrolyzing a number of plant glucosides, including L-picein (p-hydroxyacetophenone-beta-D-glucoside) and prunasin (D-mandelonitrile-beta-D-glucoside). Taking advantage of the marked differences in the spectra of the substrate/product pairs of L-picein/p-hydroxyacetophenone and prunasin/mandelonitrile, we have devised spectrophotometric assays that permit the continuous monitoring at pH 7.0 of p-hydroxyacetophenone (piceol) release from L-picein by guinea pig hepatic cytosolic beta-glucosidase and mandelonitrile from prunasin by almond beta-glucosidase. When L-picein hydrolysis was monitored at 320 nm and prunasin at 282 nm, the molar absorption coefficients determined for their products, namely piceol and mandelonitrile, were 3200 and 1360 M-1 cm-1, respectively. The kinetic parameter Km and Vmax values obtained using these spectrophotometric procedures for the guinea pig liver cytosolic beta-glucosidase acting on L-picein were 0.88 mM and 5.29 x 10(5) units/mg protein and for the almond beta-glucosidase acting on prunasin, Km 1.1 mM and Vmax 5.24 x 10(6) units/mg protein. These values agreed well with previously reported values obtained using less convenient, discontinuous assay procedures.
Lai
L B
LB
Department of Biochemistry, School of Medicine, University of New Mexico, Albuquerque 87131.
Gopalan
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V
Glew
R H
RH
D000818:Animals; D003600:Cytosol / Q000201:enzymology; D005960:Glucosides; D006168:Guinea Pigs; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D009570:Nitriles; D010944:Plants / Q000201:enzymology*; D013050:Spectrometry, Fluorescence / Q000379:methods; D013056:Spectrophotometry, Ultraviolet / Q000379:methods; D001617:beta-Glucosidase / Q000032:analysis* / Q000378:metabolism
D005960:Glucosides; D009570:Nitriles; C019063:prunasin; C050880:picein; D001617:beta-Glucosidase