How do you calculate initial velocity with time and absorbance as the variables?

Answer 1

Convert the velocity Vmax from ΔA/min to nmol/min (milliunits, mU).

For example:

1 Unit = 1 U = 1 μmol PNP formed per min

1 milli-Unit = 1 mU = 1 nmol PNP formed per min

Extinction coefficient ε for PNP at 410 nm = 15,000 M-1 cm-1

And the pathlength = 0.34 cm

if the velocity v is determined to be 0.294 ∆A/min (absorbance units per min), then:

0.294 ∆A /min * M/(15,000 A * 0.34 cm) = 0.0000196 M/min = 19.6 μM/min = = 19.6 μmol / (liter * min) = 0.0196 μmol / (ml * min)

The enzymatic reaction was carried out in 250 μl solution, hence the velocity of the enzyme is

0.0196 μmol / (ml * min) * 0.25 ml = 0.0049 μmol/min = 4.9 nmol/min PNP generated. v = 4.9 nmol/min = 4.9 mU