How do I calculate the number of thiol groups?

[Olly1996]

I did a purification protein experiment and for my report I need to determine the amount of thiol groups. For the absorbance value of standard ovalbumin, with SDS I got 0.142 and without SDS I got 0.09. I understand that with SDS the answer should be in or around 4 and without SDS it should be 0. The following information is what I’m given:

Use the equation: V x ΔA412 / ε x n
V is the volume of the sample, which is 3ml.
ΔA412 = Final ΔA412 – Initial ΔA412
ε is the molar extinction coefficient of DTNB (13.6 x 106 M-1
cm-1).
n is the molar quantity of ovalbumin in the sample (molecular weight of ovalbumin is
43 000).
The standard ovalbumin provided is (7.5 mg/ml)

If anyone could help figure how to work out the number of thiol groups. I would be much appreciated. Thank you in advance

[claudepa]

This is the way I would make the calculation. However I find something wrong in the numerical application. Is your eps OK ? I do not see why using V.

If I understand well the data your equation starts from the beer lambert equation: OD =eps x l x C where l is the length of the cuvette and C the concentration.
This gives C= OD/ eps x l C is the thiol concentration. usually l = 1 cm.
If z is the number of thiol per ovalbumin, the thiol concentration is therefore z x 7.5/43000 M
So z = C x 43000 /7.5 = OD x 43000 / eps x 7.5

OD is your delta 412, OD is optical density

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