Chlorophyllase activity assay

Dear Sravani,

Sorry for my late reply. The protocol of chlorophyllase activity assay is as below.

220 µL of reaction buffer (100 mM sodium phosphate, pH 7.0), 40 µL of acetone-dissolved chlorophylls at a final concentration of 1000 µM, and 40 µL of acetone were premixed and incubated at 40 °C for 5 min. After 100 µL of purified enzyme was added, the enzyme reaction was carried out in a shaking bath (500 rpm, Eppendorf ThermoMixer C) at 40 °C for 30 min. After that, the reaction was quenched by the addition of 4400 µl of stop buffer (10 mM KOH/acetone/n-hexane=1:4:6 (v/v)). For phase separation, the mixed solution was centrifuged at 10,000 × g for 5 min. The unreacted substrate chlorophyll a, chlorophyll b, or bacteriochlorophyll a was extracted in the upper n-hexane phase. The reaction product, chlorophyllide a, chlorophyllide b, or bacteriochlorophyllide a remained in the lower aqueous acetone solution.

The reaction product in the aqueous acetone phase was determined with a spectrophotometer using an millimolar extinction coefficient of 54.1 mM⁻¹ cm⁻¹ at 665 nm for chlorophyllide a, 42.0 mM⁻¹ cm⁻¹ at 650 nm for chlorophyllide b, or 42.1 mM⁻¹ cm⁻¹ at 773 nm for bacteriochlorophyllide a. One unit of enzyme activity was defined as the amount of enzyme that hydrolyzes the chlorophyll substrate and releases 1 nmol chlorophyllide a, chlorophyllide b, or bacteriochlorophyllide a per minute at 40 °C. All enzymatic assays were carried out in triplicate. Controls were performed at the same reaction conditions as the chlorophyllase activity assays to ensure that there was no spontaneous substrate hydrolysis, by substituting the enzyme with distilled water.

Best Regards

Sitian Gu