Enumeration of thiol groups in ovalbumin

We done a lab where we purified a sample of ovalbumin from egg-white. I need some help understanding why we done some of these processes. When we mixed ammonium sulphate with the sample of egg-white, was this to denature the proteins?

We used a reagent called DTNB(5,5'-bisdithio-2-nitrobenzoate), we mixed this with 4 samples A,B,C and D. After mixing these with each sample, we recorded the absorbance readings at 412 nm at intervals for 15 minutes.

Here I have a few questions, what does DTNB do? And what pattern should we have expected?(Don't worry too much about answering this, our spectrophotometer kept giving us negative measurements, so we didn't record any results. But we are planning to get some results from another group)

In each sample, there was ovalbumin(in samples B and D), SDS(sodium dodecyl sulphate, only in samples C and D), Tris and some water. Then the DTNB was added and the absorbance was recorded for each sample. Why was there Tris in the samples?

SDS is a chemical agent used to denature protein molecules, so this straightened out the polypeptide chain. How would this affect the readings from the spectrophotometer?

No, this is ''salting out'' the proteins you need, it's part of a purification process.



DTNB will react with the RSH groups on the amino acids in the protein. As it reacted you should have seen the byproducts formed from that reaction by spectrometry.



As in Trizma? Probably acting as a buffer



Tertiary structures of proteins include disulfide bonds which wouldn't react if the protein remained folded (they wouldn't be thiols).

Hope that helps

I guess we were in the same lab class! University of Warwick