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1 Ketoconazole is an anti-fungal drug. The structure is shown below in all three forms – acid form, zwitterionic form and basic form. (note: part of the problem is to figure out which molecule corresponds to which form!) Form C has the highest absorbance rate. The pH of the stomach is approximately 3; the upper part of the small intestines has a pH of 5. What fraction of each form of ketoconazole (A,B and C) are present at in the stomach and in the intestines? Where is ketoconazole absorbed best? Briefly explain. NOTE: this molecule has two pKas—2.94 and 6.51. 2. The value for the equilibrium constant for the reaction shown is 425. In the citric acid cycle fumarate is converted to L-malate with the help of fumarase. What is the ΔG for this reaction in the TCA cycle if the concentration of L-malate is 1 µM and fumarate is 1.5 mM? 3. I have a sample that contains three small peptides (intact sample run on lane 1 of SDS-PAGE gel). My first attempt to separate them was to use size exclusion chromatography. Two peptides (peptide 1 and peptide 2) are still running together (lane 4), but I have successfully separated the third peptide (lane 4). Your job is to: (a) Deduce the sequence of peptide 1 and peptide 2 from the digest information below. (b) Describe an experimental procedure that utilizes ion exchange chromatography that will separate peptide 1 and peptide 2 from one another. Your procedure should contain 2 steps Step 1: making 3 L of a 200 mM buffer at your desired pH. Step 2: explaining what type of ion-exchange resin you will run the column to collect your samples (c) Give appropriate descriptions for lanes 5 and 6 for the SDS-PAGE gel, according to YOUR procedure. This step is your expected result from your experimental procedure. Think carefully here!

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3.

Experimental procedure for ion-exchange chromatography
Both peptides contain arginine and lysine that make the peptide positively charged. Thus the peptides can be separated by Cation exchange chromatography using a low pH buffer ( pH = 6)
Step 1: making 3 L of a 200 mM buffer at your desired pH.
Making MES buffer:
Molar mass: 195.2 g/mol
117.12 g MES (free acid) is dissolved in 2000 mL of ddH2O
pH is adjusted to the desired value with 10 N NaOH/ HCl
Volume is then made up to 3000 ml

Step 2: explaining what type of ion-exchange resin you will run the column to collect your samples

Cation exchange resin is required to separate the peptides. At low pH and low ion concentration, peptides will attach to the column (with the negatively charged matrix). Then elution with a low salt buffer ( 400mM NaCl) will elute peptide 1 as it has low positive charge ( one R and one K amino acid). A high salt buffer (1M NaCl) will completely elute the peptide 2 as it has high positive charge (two R amino acid)....
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