Glycolate oxidase (EC1.1.3.15) is an enzyme in the glycolate cycle. It is also a key enzyme in plant
photorespiration metabolism. It can catalyze the oxidation of glycolic acid to glyoxylic acid. By measuring
the activity of glycolate oxidase, we can understand the basic methods of plant photosynthesis and
respiratory metabolism.
Glycolate oxidase catalyzes the oxidation of glycolic acid to glyoxylic acid. Glyoxylic acid reacts with
phenylhydrazine hydrochloride to form phenylhydrazone glyoxylate. There is a characteristic absorption
peak at 324 nm.
Required but Not Provided:
Ultraviolet spectrophotometer, low temperature desk centrifuge, balance, transferpettor, mortar/
homogenizer, ice, 1 mL quartz cuvette and distilled water.
Protocol
I. Preparation:
1. Tissue: according to the ratio of mass (g): Extract solution volume (mL): 1:5-10 to add the extract. It is
suggested that add 1 mL of extract to 0.1 g of tissue. Homogenate on ice. Centrifuge at 10000 g 4℃ for 10
min. Take the supernatant on ice for test.
2. Cells: according to the number of the cells (104): the volume of the Extract solution (mL) is
500~1000:1. It is suggested that add 1 mL of extraction reagent to 500 million of cells. Breaking cells by
ultrasonic wave in ice bath (power 300W, ultrasonic 3s, interval 7s, total time 3 min). Centrifuge at 10000
g 4℃ for 10 min. Take the supernatant on ice for test.
II. Determination
