Determination of thermodynamic data by microcalorimetry: the michaelis constant of glucose oxidase immobilized on various carriers

Abstract Microcalorimetry and polarography were chosen for the investigation of dissolved and immobilized enzymes. Glucose oxidase and catalase were used bound to polyacrylamide or were immobilized by attaching them to nylon fibers which had been treated with triethyloxonium-tetrafluoroborate, diaminohexane and glutaraldehyde according to MORRIS, CAMPELL and HORNBY (1975). The latter method assured that the enzymes are located in a side chain to the polyamide structure. If, and to what degree, this immobilization could have changed the properties of the enzymes was tested by determining the MICHAELIS constant of dissolved and immobilized enzyme. Polarography, in the case of the dissolved enzymes, microcalorimetry and polarography in the case of the immobilized enzymes, led to the same K m -value. Replacement of air by oxygen increased the final heat output rate, but had no influence on the K m -value. Irradiation of the immobilized enzyme decreased the heat output rate, the K m -value remained unchanged. These results prove that once a glucose oxidase molecule is damaged it looses all activity rather than remaining partially active.