Question

2. As an alternative, people developed another enzyme mediated assay to measure glucose concentration as below: Hexokinase Glucose + ATP Glucose-6-Phosphate + ADP G6PDH G6P + NAD 6-Phosphogluconate + NADH Based on the information above and what you learned so far, how exactly can you determine the glucose concentration from this method? Hint: spectrophotometrically (10 pts)

Answer 1

By using Beer's law, the concentration of glucose can be determined, as shown below.

A1 = $\epsilon$1c1l

A2 = $\epsilon$2c2l

Where A1 and A2 are the absorbances of the solutions of Glucose-6-phosphate and 6-phosphogluconate, respectively.

$\epsilon$1 and $\epsilon$2 are the molar absorptivities of the Glucose-6-phosphate and 6-phosphogluconate, respectively.

c1 and c2 are the concentrations of the solutions of Glucose-6-phosphate and 6-phosphogluconate, respectively.

'l' is the path length = 1 cm (in general).

Now, the overall absorbance (A) = A1 + A2 = ($\epsilon$1c1+$\epsilon$2c2)*l

By finding the concentrations of Glucose-6-phosphate and 6-phosphogluconate, you can find the concentration of glucose accordingly.