Determining thiol groups...??

[Dolphine]

Okay, here's my problem.

We did this purification of ovalbumin in our biochemistry labs little while ago. We need to some calculations and answer some questions concerning the practical. One question is to calculate the number of reactive thiol groups in one molecule of the protein. No matter how much I have tried I can't figure out how to do this. We were given the molecular weight of ovalbumin and we determined the absorbance of the protein at 412nm. How can I calculate the thiol groups using these bits of information? I'm getting desperate with this, the whole course is stressing me so badly. Too bad is compulsory for us. But if you can give me any tips how to solve this, please do!

Thanks very much for advance!

[sdekivit]

You tell something about reactive thiolgroups. My question is therefore: how did you do your experiment ?

I cannot calculate the amount of reactive thiolgroups from a singe absorbance.l

[Dolphine]

First we did the purification process of the ovalbumin. Then we determined the absorbances of native and purified ovalbumin, both with and without SDS and using DTNB in the samples to do it. We also took readings of the native and our sample ovalbumin at the 280nm.

[sdekivit]

First we did the purification process of the ovalbumin. Then we determined the absorbances of native and purified ovalbumin, both with and without SDS and using DTNB in the samples to do it. We also took readings of the native and our sample ovalbumin at the 280nm.

now i can do something:)

yes, now i can calculate thiolgroups What does DNTB do? Yes, it alkylates thiolgroups. And what does SDS do? yes, it keeps proteins denatured.

So what do we need to know for the amount of thiolgroups? First take the purified sample of ovalbumine and measure the absorbance. Than incubate with DNTB and SDS to denature the ovalbumine and you again measure he absorbance.

--> the difference in absorbance is the amount of thiolgroups that can be calculated with the law of Lambert-Beer.

The amount of protein is measured at 280 nm, where eacht unit of absorbance is 1 mg/ml protein.

[ros]

My name is Dr. Ros Dickison. I determined the number of thio groups in ovalbumin as part of my Masters thesis. I continued the study and in my doctoral dissertation found that when ovalbumin is dephosphorylated the numer of thio groups change indicating a conformational change. My e-mail is doctorros@msn.com

[sdekivit]

My name is Dr. Ros Dickison. I determined the number of thio groups in ovalbumin as part of my Masters thesis. I continued the study and in my doctoral dissertation found that when ovalbumin is dephosphorylated the numer of thio groups change indicating a conformational change. My e-mail is doctorros@msn.com

interesting you already examined what causes the conformational change. I'm curious