You have been provided a mixture containing four amino acids (Glu, His, Pro and Lys) to be separated by ion-exchange chromatography using a Dowex 50 cation-exchange, prepared in a loading buffer at pH 2.0. The following solutions are made available:

• The amino acid mixture in 0.04M citrate buffer at pH 2.0.

• 0.04M citrate buffer, pH 4.5

• 0.04M citrate buffer, pH 8.0

• 0.04M citrate buffer, pH 10.5

  Is complete resolution of all amino acids achieved if the buffers are allowed to elute through the column sequentially from lowest to highest pH? Identify the order of elution and identify any amino acids that may come off the column together if complete resolution is not achieved. Support your response with an explanation.
  Assuming the amino acids in the mixture will elute from the column ONLY at a solution pH that is above their respective pI.

1. An anion-exchange resin can be used instead of the cation-exchange resin in this experiment to separate the three amino acids in the known mixture. What changes to experimental protocol are required to achieve complete resolution? Support your response with an explanation. (HINT: consider pH).

2. You have been provided a mixture containing four amino acids (Glu, His, Pro and Lys) to be separated by ion-exchange chromatography using a Dowex 50 cation-exchange, prepared in a loading buffer at pH 2.0. The following solutions are made available:

3. The amino acid mixture in 0.04M citrate buffer at pH 2.0.

4. 0.04M citrate buffer, pH 4.5

5. 0.04M citrate buffer, pH 8.0

6. 0.04M citrate buffer, pH 10.5

   Is complete resolution of all amino acids achieved if the buffers are allowed to elute through the column sequentially from lowest to highest pH? Identify the order of elution and identify any amino acids that may come off the column together if complete resolution is not achieved. Support your response with an explanation.
   Assuming the amino acids in the mixture will elute from the column ONLY at a solution pH that is above their respective pI.

2. Describe the mechanism by which muscle tissue uses branched-chain amino acids as fuel and passes the nitrogen from these amino acids to liver tissue to be converted to urea.

Please offer detailed explanations. Thank you.

1. What is the turnover number? How would you calculate it?

2. Which amino acids are small and hydrophobic? Large and hydrophobic? What type of binding site (meaning what amino acids would it contain) would bin a negatively charged amino acid? A positively charged one? A small hydrophobic one? A large hydrophobic one?

1. we discussed a classification scheme of amino acids that divided them into three groups based on Polarity. what are the three groups of amino acids and where are you most likely to find amino acid residues belonging to each group within a protein?

2. what are the benefits and pitfalls of each method of protein structure determination?

1.     Oxaloacetate can be converted into several different metabolic compounds. Briefly discuss each one, and include whether that is part of a catabolic or anabolic pathway.

2.     There are several allosteric regulators of pyruvate carboxylase. For each one, explain the following:

o   Is this molecule an allosteric activator or allosteric inhibitor?

o   Why does it make sense that it would regulate the enzyme in that way?

o   Does this inhibitor/activator stabilize the R state or T state of the enzyme?

Acetyl CoA

Aspartate

3.     Draw a Michaelis Menten graph to represent normal pyruvate carboxylase kinetics. Explain why you drew things as you did.

A deficiency in pyruvate carboxylase is a rare autosomal recessive inborn error in metabolism. The most severe form is called Type B or French phenotype

4. Explain why a deficiency in pyruvate carboxylase would result in the following metabolic issues:

o   State the specific pathway(s) that produces the condition. For example, answer glycolysis instead of just glucose metabolism.

o   Explain why this pathway would be involved during pyruvate carboxylase deficiency.

a.     Lactic acidemia, with an elevated lactate to pyruvate ratio

b.     Ketoacidosis

c.      Hypoglycemia

d.     Hyperammonemia

e.     Hypercitrullinemia

A milder form of the disease is Type A. These individuals only have mild lactic acidemia and psychomotor retardation and live longer, though some die within a few years. One of the mutations that causes Type A is a mutation from Alanine to Threonine at amino acid 610, located within the pyruvate binding domain

5.     Draw the predominant form of the amino acids alanine and threonine at the following pH values.

pH 7: alanine and threonine

pH 4: alanine and threonine

6.     What non-covalent interactions can each amino acid participate in, as part of tertiary structure at pH 7?

Suggest one amino acid whose side chain can participate in a non-covalent interaction with the side chain of alanine, and one amino acid whose side chain can participate in a non-covalent interaction with the side chain of threonine.

Include an explanation of the charges involved in the interaction: full or partial charges? Are the charges permanent or temporary?

Alanine

Threonine

7.     Let’s assume that this amino acid is on the surface of the protein. Would you expect this mutation (alanine mutated to threonine), to have any effect on the three-dimensional shape of the protein? Explain your reasoning.

8.     What is your topic of choice? A topic could be a metabolic pathway, a disease, an enzyme, a biochemical principle (ex. hydrophobic effect, enzyme properties), a thermodynamic principle (ex. Entropy), etc.

a.      What is the topic and why did you choose this topic?

To learn more about the topic, consult your textbook and also find 2 references (primary or secondary) published in the last 10 years and write a paragraph. Cite your sources.

b.     Why would this topic be of interest to non-science majors? Explain how would you teach a key idea to someone who does not have a science background. Your answer should include what you would actually say to that person. You may include images (cite sources)

c.      Suppose you have a friend who is a science major and must take this course next semester. How would you explain a key idea of your topic to that friend? Your answer should include what you would actually say to that friend. Images can be posted too.

Question 8.  A transmembrane protein uses a β barrel structure to span the cell membrane.  How can amino acid mutations in a protein within this β barrel structure affect interactions with the membrane and why? (Up to 50 words)  

Question 9.  Haemoglobin is a protein that carries oxygen in our red blood cells.  Sickle cell anaemia is a genetic disease in which Glu at position 6 of haemoglobin is mutated to a Val, rendering haemoglobin non-functional.

1. What type of amino acid change is this?  (1 mark)

2. Give one reason why this could affect the function of this protein.  (1 mark)

Question 10.  Lectin is a protein that binds carbohydrates and is critical for biological recognition processes in living organisms.  Describe what bonds and forces could be involved in lectin and carbohydrate interactions. (Up to 50 words)  

Question 11.  A hexokinase is an enzyme that adds a phosphate to glucose after it enters the cell, which is considered the first step of glycolysis.  One enzyme, hexokinase A (HKA), has a K_m of 0.02 mM, whereas another, hexokinase B (HKB), has a K_m of 1.0 mM.  Explain why some types of fast growing cancer cells would use HKA instead of HKB. (Up to 50 words)  

how do buffer ph/pKa/pI values for amino acids affect their elution order during ion exchange chromatography?

what molecular properties affect the migration distance (on the chromatography paper) of amino acids during IEC?