A novel protein of unknown structure has been purified. Using chromatography, th

Question

A novel protein of unknown structure has been purified. Using chromatography, the native molecular weight is determined to be 240,000. Chromatography in the presence of 6M guanidine results in a protein with an apparent molecular weight of 60,000. Chromatography in the presence of both 6M guanidine and 10 mM beta-mercaptoethanol results in two polypeptides of molecular weights equaling 34,000 and 26,000. What does this experiment tell you about the subunit composition of this protein? It may be helpful if you sketch a cartoon of the protein.

Explanation / Answer

ANswer: Guanidine hydrochloride is a powerful denaturing agent . Ths chemical destroys both the quaternary and tertiary structure of protein by breaking hydrogen bonds. when 6M guanidine-HCl is used during chromatography, molecular weight of given protein was observed to be 60,000. This indicates that the native protein might be basically be a tetramer of four subunits each having molecular weight of 60,000 dalton. when protein was denatured in the presence of ?-mercaptoethanol, two protein peaks are observed, one at 34,000 dalton and one at 26000Dalton. since Beta- mercaptoethanol is a disulfide reducing agent, it will reduce or break apart disulfide bonds. It can be said that 60,000 dalton subunit is a heterodimer of two chains, 34,000 Dalton and 26,000 Dalton. These two chains are held together by a disulfide bond.

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