For the proteins listed below indicate how you would effectively separate each p
ID: 202998 • Letter: F
Question
For the proteins listed below indicate how you would effectively separate each protein from the other proteins in that mix by answering the questions. Protein name Function pl based on amino acid sequence itongodb bn Size based on amino acid sequence (in kDa) 215 210 7.5 Pro1P Flxip 7.3 120 5.4 Hmmip Cncip Binds to AMP and Ca* Protein kinase, catalyzes the reaction between a protein and ATP to give ADP and a phosphorylated protein Binds to iron (Fe**) Protein phosphatase, catalyzes the reaction that removes a phosphate from a phosphorylated protein Unknown but does bind to Zinc metal ions 35 8.8 Unkp 7.8Explanation / Answer
Question a.
Answer. Ion exchange Chromatography can be used to separate these proteins.
If we take a buffer having PH 7.0 and dissolve all these proteins into them then all the proteins will have positive charge except Hmm1p [ PI 5.4 ; it will be negatively charged].
Always remember it as a thumb rule when ;
PH < PI - Protein will have positive charge.
PH > PI - Protein will have negative charge.
As most of our protein has postive charge( except one) we will use "Cation exchange resin" that is the resin having negative charge. e.g. Carboxymethyl.
Order of elution from the chromatography column : Hmm1p > ~ flx1p> ~ Pro1p > Unk1P > Cnc1p.
Question b.
Answer. Change in pH can have an effect of the state of ionization of acidic or basic amino acids. Acidic amino acids have carboxyl functional groups in their side chains. Basic amino acids have amine functional groups in their side chains. If the state of ionization of amino acids in a protein is altered then the ionic bonds that help to determine the 3-D shape of the protein can be altered. This can lead to altered protein recognition or an enzyme might become inactive.
Question(c).
Answer. Buffer exchange is one of the possibility to regain the 3-D stucture or elute the proetin in a buffer having PH which is also the optimum PH for that enzyme.