Abnormal inhibitory activity problem ( ) – ( ) X100 = 120 (%) !? ( ) – ( ) X100 = -10 (%) !!??

1.3 Abs. valid region (linear) observed theoretical conc. 0.4 mechanical limit most accurate point Use of absorbance below 1.3 is recommended in any measurements. Lower absorbance than this upper limit is assumed to be proportional to concentration. invalid region (non-linear)

1.3 Abs. conc Liberated glucose control test ( ) – ( ) X100 = 30 (%) 0.1 observed theoretical Normal case (no problem) blank test control

1.3 Abs. observed theoretical conc Liberated glucose control test (apparent) 0.1 apparent test (true) 2.0 true High blank case (due to inherent glucose or pigment) 1.0 two-fold dilution observed (100%) (70%) (50%) x ( ) – ( ) X100 = 50 (%) Apparent inhibition ( ) – (1.0x2-1.3) X100 = 30 (%) Actual inhibition

1.3 Abs. conc Liberated glucose control test ( ) – ( ) X100 = 30 (%) 0.1 observed theoretical Inherent sucrose problem blank test control ( ) – ( ) X100 = 20 (%) additional glucose from sucrose in sample Apparent inhibition Actual inhibition % inhibition would become minus

e.g. Strawberry the sweetest variety contains 40 mg sucrose/ g fr. wt. Sucrase assay solution 56 x x 0.2 = 3.8 (mg) 0.2 mL of 56mM sucrose 0.1 mL of 1n sample solution x 0.1 = 0.4 (mg) X 100 = 11(%) sucrose concentration increase

Samples likely to contain a higher amount of sucrose than strawberry, banana, sugar cane and so on should be neglected to evaluate an anti-sucrase activity. Does anyone have good idea about it? Safer guideline If absorbance of sample or sample blank exceeds 1.2, then dilute it so as to become smaller than 1.2. Multiply the absorbance after dilution by the dilution factor. e.g. Original Abs Abs. after 2 times dilution Corrected Abs. would be 0.800x2=1.600