1. Separation of amino acids by ion-exchange Chromatography. Mixtures of amino a
ID: 35630 • Letter: 1
Question
1. Separation of amino acids by ion-exchange Chromatography. Mixtures of amino acids can be analyzed by first separating the mixture into its components through ion-exchange chromatography. Amino acids placed on a cation-exchange resin containing sulfate (-SO3-) groups flow down the column at different rates because of two factors that influence their movement (1) ionic attraction between the sulfonate residues on the column and positively charged functional groups on the amino acids, and (2) hydrophobic interactions between amino acid side chains and the strongly hydrophobic backbone of the polystyrene resin. For each pair of amino acids listed, determine which will be eluted first from the cation-exchange column by a ph 7.0 buffer.
a.) Asp and Lys
b.) Arg and Met
c.) Glu and Val
d.) Gly and Val
e.) Ser and Ala
2. Note the structure of the heptapeptide:
NH2-ser-asp-gln-tyr-gly-cys-thr-COOH.
Would you expect the peptide to dissolve well in water?
Answer the same for the heptapeptide:
NH2-trp-ala-gly-met-val-leu-phe-COOH.
3. At pH=7, do you expect the following peptides to bind a cation exchanger or an anion exchanger resins?
a) NH2-ser-glu-cys-tyr-ala-asp-lys-COOH
b) NH2-ala-arg-lys-thr-asn-glu-lys-COOH
Explain.
4. List four different proteases that are used in peptide mapping, and provide their cleavage specificity.
5. You have a solution in your hand that contains your protein. You want to sequence the protein. Use the PPT presentation included in Topic 5B to make a step by step list of how this process is carried out.
6. The following polypeptide was digested with the proteolytic enzyme trypsin.
NH2-Met-Gln-Tyr-Thr-Ser-Gly-Asn-Arg-Trp-Ala-Gly-Val-Leu-Cys-Pro-Lys-Gln-Glu-Asp-Ser-Asp-Glu-Tyr-Thr-COOH
A. Write the resulting peptide fragments and mark the amino acids whos side chains are expected to be charged at pH=7 (Write + or - charge)
B. You are asked to separate the peptides from one another by a two-step column chromatography procedure. At your disposal are ion exchange columns (cation or anion, based on your choosing), and a hydrophobic column. Describe how you would carry out chromatography to accomplish this separation. Your description should explain how the various fragments separate from each other in each step.
Explanation / Answer
pI Values
Net charge (pH 7)
Elution order
Basis for separation
Asp, Lys
2.8, 9.7
-1, +1
Asp, Lys
Charge
Arg, Met
10.8, 5.7
+1, 0
Met, Arg
Charge
Glu, Val
3.2, 6.0
-1,0
Glu, Val
Charge
Gly, Leu
5.97, 5.98
0,0
Gly, Leu
Polarity
Ser, Ala
5.68, 6.01
0,0
Ser, Ala
Polarity
2)Heptapeptide: NH2-ser-asp-gln-tyr-gly-cys-thr-COOH. The extinction coefficient is 1280 M-1cm-1. Iso electric point is 0.75 and Net charge at pH 7. This heptapeptide will show good water solubility.
Heptapeptide: NH2-trp-ala-gly-met-val-leu-phe-COOH. The extinction coefficient is 5690 M-1cm-1 and Iso-electric point is 3.58 and Net charge at pH 7 is 0. This peptide will show poor water solubility.
Polar amino acids with no charge on R group are soluble in water (hydrophilic); they can participate in hydrogen bonding of with water.
3) NH2-ser-glu-cys-tyr-ala-asp-lys-COOH
NH2
ser
glu
cys
tyr
ala
asp
lys
COOH
Net charge
+1
0
-1
0
0
0
-1
+1
-1
-1
Peptide will bind to the anion exchanger. In anion exchange resin, proteins that are negatively charged at the loading buffer pH will bind to the positively charged column resin.
4)
Staphylococcus V8 protease
cleaves peptide bonds on the carboxy terminal side of either aspartic or glutamic acids
Trypsin
C-terminal side of Arg and Lys
Clostripain
C-terminal side of Arg
Chymostrypsin
C-terminal side of hydrophobic residues (e.g. Leu, Met, Ala, aromatics)
pI Values
Net charge (pH 7)
Elution order
Basis for separation
Asp, Lys
2.8, 9.7
-1, +1
Asp, Lys
Charge
Arg, Met
10.8, 5.7
+1, 0
Met, Arg
Charge
Glu, Val
3.2, 6.0
-1,0
Glu, Val
Charge
Gly, Leu
5.97, 5.98
0,0
Gly, Leu
Polarity
Ser, Ala
5.68, 6.01
0,0
Ser, Ala
Polarity
Related Questions
Navigate
Integrity-first tutoring: explanations and feedback only — we do not complete graded work. Learn more.