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Glucose determination using immobilized polyphosphate glucokinase.

T H Kowalczyk1, O Szymona

  • 1Department of Biochemistry, School of Medicine, Lublin, Poland.

Analytical Biochemistry
|September 2, 1991
PubMed
Summary
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Immobilized polyphosphate glucokinase on silica gel beads offers a stable and specific method for glucose determination. This reusable enzyme reactor provides accurate and precise results for serum glucose analysis.

Area of Science:

  • Biochemistry
  • Enzyme immobilization
  • Analytical chemistry

Background:

  • Traditional glucose determination methods often rely on ATP, which can be less stable than polyphosphate.
  • Hexokinase, a common enzyme for glucose assays, exhibits lower specificity compared to polyphosphate glucokinase.
  • Enzyme immobilization enhances enzyme stability, reusability, and can simplify assay procedures.

Purpose of the Study:

  • To develop and characterize an immobilized enzyme reactor for glucose determination using polyphosphate glucokinase.
  • To evaluate the stability, specificity, and reusability of the immobilized enzyme for practical applications.
  • To compare the performance of the immobilized enzyme assay with soluble enzyme methods.

Main Methods:

  • Covalent coupling of polyphosphate glucokinase to collagen-coated silica gel beads.

Related Experiment Videos

  • Construction of a packed-bed reactor using the immobilized enzyme.
  • Spectrophotometric measurement of NADPH produced in coupled enzymatic reactions for glucose quantification.

    • Main Results:

    • The immobilized enzyme reactor demonstrated good linearity for glucose concentrations between 10 and 50 nmol.
    • High precision (CV < 4%) and accuracy (approx. 100% recovery) were achieved.
    • The reactor exhibited good operational stability over a month, with only a 12% loss in activity.
    • Excellent correlation (r = 0.997) was observed with soluble polyphosphate glucokinase assays.

    Conclusions:

    • Immobilized polyphosphate glucokinase provides a robust, specific, and reusable platform for glucose determination.
    • The developed method offers advantages in enzyme stability and specificity over traditional hexokinase-based assays.
    • The immobilized enzyme reactor is suitable for routine glucose analysis in biological samples like serum.