| Enzyme | % Yield of HK Activity (expressed | Specific Activity | Fold Purifica
ID: 206263 • Letter: #
Question
| Enzyme | % Yield of HK Activity (expressed | Specific Activity | Fold Purification Total protein (mg) (umol/min mg (expresse relative to | relative to (umol/min) 406 146 5075 100 Crude extract Ammonium sulfate 1540 216 chromatography98 Gel fitration89 0.42 37 Affinity chromatography As part of the gel filtration run, you calibrated the elution volume with proteins of known molecular weight and generated graph A. The HK activity eluted at 60 ml. You subsequently ran gel electrophoresis of the purified protein in the presence of SDS with and without the reducing agent, mercaptoethanol. The gel included proteins of known molecular weight, and an image of the gel is shown in B. Using information from gel filtration and gel electrophoresis, determine the native molecular weight, the subunit composition of the intact protein (number and size of subunits), and the nature of the interactions between subunits in the native protein. Mw Sample A. 3. kD 80 60 |- 40 54 5.2 5.0 4.X 5 30 2o 40 Lo Bo 100 Vol 6Explanation / Answer
Molecular weight of native protein is app. 50kd.
There 2subunits dimeric protein.
These 2 subunits are bonded by disulfide bridge. Because b-mercaptoethanol particularly acts on disulfide bridges and breaks them.so here 2 subunits of 30 of are bonded by disulfide bonds and forms dimer of 50kd.
Disulfide bonds between two cystein is a type of covalent bond that makes protein more stable. Apart from these , some noncovalent interaction like Vander Waal forces, hydrogen bond, also involve in the interaction of monomers that makes protein stable structure.
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