Question

2. Two different experiments were performed to determine the quatemary structure of HCV reverse transcriptase as well as the molecular size of each subunit. In these experiments, HCV reverse transcriptase was mixed with equal masses of two proteins with known molecular weights of 10 KDa and 80 KDa. Both of these proteins consisted of a single polypeptide chain. This mixture was then separated by gel filtration (below) or by SDs-PAGE (below) Gel Filtration Column: The absorption as a function of the elution volume is shown to the right. The 1st peak is the HCV reverse transcriptase. SDS-PAGE. The SDS gel is shown on the right (turned sideways). The bands indicate regions of the gel that are stained with a stain for protein. The thicker the band, the more protein. The arrow marks the top of the gel (where the mixture of proteins would be applied to the gel before the electric field would be turned on). The lower scale gives the distance from the starting point Please answer the following questions: i) Mark the location of the anode (+ electrode) and the cathode (-ellectrode) on the SDS-gel. ii) Determine the molecular weight(s) of the polypeptide chain(s) that is (are) present in this enzyme. Be sure to show your work ili) Determine the native molecular weight of the HCV reverse transcriptase from the gel filtration data. Please show your work. Your answer should include a discussion of the quatenary structure of this enzyme (e.g. homodimer, heterodimer, trimer, etc)? -LLA 10 Elutien Vebume (mt) zo Distance fmaak

Two different experiments were performed to determine the quaternary structure of HCV reverse transcriptase as well as the molecular size of each subunit. In these experiments, HCV reverse transcriptase was mixed with equal masses of two proteins with known molecular weights of 10 KDa and 80 KDa. Both of these proteins consisted of a
single polypeptide chain. This mixture was then separated by gel filtration (below) or by SDS-PAGE (below).
Gel Filtration Column: The absorption as a function of the elution volume is shown to the right. The 1st peak is the HCV reverse transcriptase.
SDS-PAGE. The SDS gel is shown on the right (turned sideways). The bands indicate regions of the gel that are stained with a stain for protein. The thicker the band, the more protein. The arrow marks the top of the gel (where the mixture of proteins would be applied to the gel before the electric field would be turned on). The lower scale gives the distance from the starting point.
Please answer the following questions:
i) Mark the location of the anode (+ electrode) and the cathode (- electrode) on the SDS-gel.
ii) Determine the molecular weight(s) of the polypeptide chain(s) that is (are) present in this enzyme. Be sure to show your work.
iii) Determine the native molecular weight of the HCV reverse transcriptase from the gel filtration data. Please show your work. Your answer should include a discussion of the quaternary structure of this enzyme (e.g. homodimer, heterodimer, trimer, etc)?

WOULD YOU PLEASE EXPLAIN THESE ANSWERS ? THANK YOU

Answer 1

Answer i:-

1. When the proteins are subjected to electrophoresis, they are being treated prior with Sodium Dodecyl Sulfate which provides a negative charge to the overall protein molecule. So they will migrate from the cathode to the anode.
2. So, the protein subunits will be present at the 0 position on scale which can be considered as the cathode or the negatively charged electrode.
3. They will migrate towards the positive electrode mentioned towards the 80mm mark on the scale also termed as the anode.

Answer 2:-

1. As already mentioned in the statement above, there are two additional proteins with molecular weight of around 10 and 80kDa respectively. However, there needs to be mentioning of the control which will help to elaborate the results. Even thought the Molecular weights are provided for two additional proteins, the distances for them are not mentioned. The Rf value needs to be calculated which needs to be marked by using a dye for molecular weight determination.
2. Log of respective molecular weight is taken as 4.38 (80kDa) and 2.30 (10kDa).
3. This can be used to plot a graph vs the Rf value in order to estimate molecular weight of unknown.
4. Additional data is needed.