Question

Calculate the concentrations of ATP and NADPH in solution with absorbances ( in 1 cm cuvette) of a) 0.15 at 340 nm and 0.90 at 260 nm, b) zero at 340 nm and 0.531 at 260 nm. The ε at 260 for NADPH is 15 mM-1 cm-1 and at 340 nm 6.22 mM-1 cm-1. For ATP at 260 nm the ε is 15.4 mM-1 cm-1 and zero at 340 nm.

Answer 1

Absorbance (A) = ecl

e = absorbitivity

c = concentration

l = path length

(A) Concentration of ATP (at 340nm)

0.15 = 0 X C X 1

C = 0

at 260nm

0.9 = 15.4 X C X 1

C = 0.9 / 15.4 = 0.058mM

Concentration of NADH+ at 340nm

0.15 = 6.22 X C X 1

C = 0.024mM

at 260nm

0.9 = 15 X C X1

C = 0.06mM

(B) Concentration of ATP

at 340 nm

0 = eCl

C = 0

at 260nm

0.531 = 15.4 X C X1

C = 0.035mM

Concentration of NADH

at 340nm

0 = eCl

C = 0

at 260nm

0.531 = 15 X C X1

C = 0.0354mM