A solution containing NAD+ and NADH has an optical density of 0.233 at...
Solution to determine the concentration of NAD in mMGiven information:
- Optical density at 340 nm = 0.233
- Optical density at 260 nm = 1.000
- Extinction coefficient for NADH at 340 nm = 6220 M^-1cm^-1
- Extinction coefficient for NAD at 260 nm = 18000 M^-1cm^-1
Step 1: Calculate the concentration of NADH in the solution using Beer-Lambert law.
Beer-Lambert law states that the absorbance (A) of a solution is directly proportional to the path length (l) of the light through the solution, the concentration (c) of the absorbing species and the molar extinction coefficient (e).
A = ecl
Using the given information:
A = 0.233, e = 6220 M^-1cm^-1, l = 1 cm
Therefore, c(NADH) = A / (ecl) = 0.233 / (6220 x 1) = 3.74 x 10^-5 M
Step 2: Calculate the concentration of NAD in the solution using the difference in absorbance at 260 nm and the extinction coefficient of NAD at 260 nm.
The difference in absorbance at 260 nm is due to the presence of both NAD and NADH in the solution. Since NADH does not absorb at 260 nm, the absorbance at this wavelength is due to NAD alone.
Using the given information:
A(NAD) = A(260 nm) - A(NADH) = 1.000 - 0.233 = 0.767
e(NAD) = 18000 M^-1cm^-1, l = 1 cm
Therefore, c(NAD) = A / (ecl) = 0.767 / (18000 x 1) = 4.26 x 10^-5 M
Step 3: Calculate the concentration of NAD in mM.
Since the molar concentrations of NAD and NADH have been calculated, we can convert them to mM by multiplying by 1000.
c(NAD) = 4.26 x 10^-5 M x 1000 = 0.0426 mM
Therefore, the concentration of NAD in mM is 25.