Catalase Essay

3.6.4 Assay of Catalase (CAT)

Catalase activity was assayed by measuring the inhibition rate of Hydrogen peroxide at 240nm according to the method described by Luck (1974).

For this assay,

• A 20% homogenate of the leaf extracts of different plants was prepared in phosphate buffer, 0.067 M (pH 7.0). The homogenate was then centrifuged. The supernatant was then used as enzyme extract.

• Hydrogen peroxide (H2O2, 2mM) in phosphate buffer (3.0ml) was taken in an experimental cuvette, followed by the rapid addition of 40μl of enzyme extract and mixed thoroughly.

• The time required for a decrease in absorbance by 0.05 units was recorded at 240nm in a spectrophotometer (Genesys 10-S, USA).

• The enzyme solution containing H2O2-free phosphate buffer

The inoculum was prepared from fresh overnight broth culture in nutrient dextrose broth. Plates were incubated for 24 hours at 37°C and 96 hours at 28°C for antibacterial and antifungal activity respectively.

3.9 Chromatographic profiling and SDS-PAGE of Peptide/protein extracted from aqueous leaf extract

The methods used sequentially for purification of aqueous extract were i) Ammonium Sulfate Precipitation, ii) dialysis and iii) Ion-exchange chromatography on DEAE-Cellulose columns.

All steps were carried out at 4ºC to maintain the stability of the isolated products unless mentioned otherwise.

The chemicals and dialysis membrane matrix used for the partial purification were ammonium Sulfate, Disodium Hydrogen Phosphate, Sodium Hydrogen Phosphate, Sodium Chloride, dialysis membrane cut off 5 kDa and DEAE-Cellulose. All were obtained from Himedia Labs.

3.9.1 Ammonium sulfate fractionation

Aqueous extracts of the M. oleifera were precipitated with solid ammonium sulfate (NH2SO4) to isolate the proteins into different fractions, using varying degrees of saturation. Increasing degrees of saturation with ammonium sulfate were used. Saturation ranges of 0-30%, 30-50%, and 50-80% were initially used for aqueous