Monitoring carbohydrate enzymatic reactions by quantitative in vitro microdialysis.
On-line in vitro microdialysis (MD) sampling followed by HPLC separation and UV absorbance detection (HPLC-UV) was used to monitor carbohydrate enzyme systems. Fundamental parameters (i.e., K(m) and V(max)) of hydrolysis reactions of 4-nitrophenyl-beta-D-glucopyranoside, 4-nitrophenyl-beta-d-galactopyranoside, and 4-nitrophenyl-beta-D-xylopyranoside were determined for a model enzyme, almond beta-glucosidase. Accurate quantitation was achieved via internal standard methodology and compared to spectrophotometric data and literature K(m) values, which were found to be 2.6+/-0.5 mM (MD), 2.7+/-0.4 mM (spec), and 2.5 mM (lit), for the substrate 4-nitrophenyl-beta-d-glucopyranoside. A previously unpublished K(m) value for the substrate salicin was also determined by this method. An application is shown for monitoring the glycoside salicin and its hydrolysis product saligenin in a commercially available willow bark product that is used for making tea. This versatile method has far-reaching applications to monitoring a variety of carbohydrates in enzymatic processes without complex sample preparation procedures and without volume loss.
Modi
Swati J
SJ
Department of Chemistry and Biochemistry, University of Maryland, Baltimore County, 1000 Hilltop Circle, Baltimore, MD 21250, USA.
LaCourse
William R
WR
D001592:Benzyl Alcohols / Q000737:chemistry / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid / Q000295:instrumentation / Q000379:methods; D015314:Dialysis Solutions / Q000032:analysis* / Q000737:chemistry; D004798:Enzymes / Q000378:metabolism; D005960:Glucosides; D006027:Glycosides / Q000737:chemistry / Q000378:metabolism*; D006868:Hydrolysis; D007700:Kinetics; D017551:Microdialysis / Q000295:instrumentation* / Q000379:methods; D015394:Molecular Structure; D024301:Plant Bark / Q000737:chemistry; D015203:Reproducibility of Results; D032108:Salix / Q000737:chemistry; D013056:Spectrophotometry, Ultraviolet / Q000295:instrumentation / Q000379:methods; D001617:beta-Glucosidase / Q000378:metabolism*
D001592:Benzyl Alcohols; D015314:Dialysis Solutions; D004798:Enzymes; D005960:Glucosides; D006027:Glycosides; C005696:salicin; D001617:beta-Glucosidase; C010631:salicyl alcohol