Calculate the concentration for a sample of Cyt C with an absorbance at 280nm of 6 , giving your answers in units of mg protein/ml

Answer 1

Absorbance at 280 nm is a quick way for measuring concentration of a protein (if the protein contains aromatic amino acids such as tryp, phe and try).

Use the Beer-Lambert's Law:

A=e.c.l (A=absorbance; e =extinction coefficient and l=cuvet path length)

In your problem, e and l are not given. We often use l=1cm since most cuvets we use in the lab have a path length of 1 cm. Cytochrome c is a red-colored protein (since it contains the heme group just like hemoglobin). Most proteins are not visibly colored since amino acids do not absorb light in the visible wavelength. Let us assume the cytochrome c is from horse heart (it is not specified in your problem). You will therefore find values of e in literature at different wavelengths (e.g., 550 nm and 280 nm for cytochrome c). You must use the value at 280 nm since your problem measured the absorbance at 280 nm.

I got one from literature of:

e at 280 nm cytochrome c horse heart = 0.26 x 10⁵/M.cm (note the unit is in Molar, M)

l=1 cm

c=concentration

A = e.c.l

6 = 0.26x10⁵.c.1

c = 6/0.26x10⁵

c= 2.3 x 10^-4 M

= 2.3 x 10^-4 mol/L

The molecular weight of horse heart cytochrome c = 12,384

We must convert c from mol/L into g/L

= 2.3 x 10^-4 x 12384

= 2.85 g/L. (Note that M = mol/L; to convert into g/L, you multiply by molecular weight)

= 2.85 mg/mL