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Using DTNP to find free thiol groups on a protein

Using DTNP to find free thiol groups on a protein


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I've been tasked with using DTNB to find the number of thiol groups on a molecule of Bovine Serum albumin (BSA).

After measuring the absorbance, finding the concentration of TNB and calculating the concentration of the BSA, I've come out with a result of around 0.4 free thiol groups per molecule of BSA, which seems unlikely! There is either a free group or there isn't!

Yet, the implication from the demonstrator is that is is the correct result!

How can this be? Is it possible to get a value of less than one in an experiment like this?


This is a perfectly reasonable result. Remember that you are measuring an average value for a very large population of BSA molecules. Essentially what the 0.4 means is that at any one time, about 40% of the BSA molecules have a free thiol, while the rest of the cysteine residues are disulfide-bonded.