Assay of glucose oxidase and catalase

Assay of glucose oxidase and catalase GOase activity 

On oxidation, homovanillic acid, HVA (4-hydroxy-3-methoxy-phenyl-acetic acid), is converted to a highly fluorescent, stable compound with an excitation wavelength of 315 nm and an emission wavelength of 425 nm. Oxidized HVA is produced in an amount proportional to that of GOase between 0.001 and 0.25 U/ml (Guil-bault, 1968). Guilbault (1976) showed that p-hydroxyphenylacetic acid has some advantages over HVA with respect to cost and fluorescence coefficient (fluorescence/concentration) and 0.01 U of enzyme could be assayed. 

GOase activity is conventionally assayed in a Warburg apparatus to measure the uptake of oxygen (Guilbault, 1976). Electrochemical methods (potentiometric or amperometric) are less sensitive than the fluorimetric methods, but are well-suited for automation (Pardue et al., 1964 Blaedel and Olson, 1964). 

The spectrophotometric assay is based on a POase indicator reaction to measure the amount of H2O2 liberated. For this reason an excess of POase and H-donor (Section 10.2.1.4.2) has to be used. One unit (U) is then expressed as the amount of enzyme liberating 1 pmole of H2O2 per min at 25°C. 

In the spectrophotometric assay system (Bergmeyer et al., 1974 Table 10.17), the total reaction mixture (3 ml) should contain about 0.5 U GOase (about 2 pg active enzyme) per ml (at 25°C), added 

---