ERT 106 Biochemistry
Assignment 4
1. Consider the following peptide:
Gly – Ile – Glu – Trp – Pro – Tyr – Gln – Phe – Arg – Lys
What amino acids and peptides are produced when the above peptide is treated with each
of the following reagents? Explain your answer in brief.
(i)
Carboxypeptidase
(ii)
Chymotrypsin
(iii)
Trypsin
(iv)
2,4-dinitro-1-fluorabenzene (DNFB)
Answer:
i)
-
ii)
-
iii)
-
Carboxypeptidase
Carboxypeptidase cleaves at the carboxyl end of peptides.
The products are : Gly – Ile – Glu – Trp – Pro – Tyr- Gln – Phe – Arg and
Lys
Chymotrypsin
Chymotrpsin cleaves peptide bonds in which aromatic amino acids (i.e., Phe, Tyr and Trp)
contribute a carboxyl group.
The products are : Gly – Ile – Glu – Trp,
Thr – Pro – Tyr,
Gln – Phe and
Arg – Lys
Trysin
Trypsin cleaves at the carboxyl end of lysine and arginine.
The products are : Gly – Ile – Glu – Trp – Thr – Pro – Tyr- Gln – Phe – Arg and
Lys
iv)
-
2,4-dinitro-1-fluorabenzene (DNFB)
DNFB tags the amino terminal amino acid.
The product is : DNP - Gly – Ile – Glu – trp – Thr – Pro – Tyr- Gln – Phe – Arg – Lys
Hydrolysis then cleaves all peptide bonds. DNP-Gly can the be identified by
chromatographic method.
2. All enzymes are named according to the classification system designed by the Enzyme
Commission (EC) of the International Union of Pure and Applied Chemistry (IUPAC) and
based on the type of reaction they catalyze. Explain all the classes with an example of the
reaction that they catalyze.
 Oxidoreductases. catalyze oxidation-reduction reactions. Subclasses of this group include
oxidases, reductases & peroxidases.
Exp : ethanol + NAD  Formaldehyde + NADH
 Transferases. catalyze the transfer of groups (carboxyl, carbonyl, phosphoryl) from one
molecule to another.
Exp : Glucose + ATP  Glucose-6- phosphate + ADP
 Hydrolases. catalyze the cleavage of bonds due to addition of water.
Exp : Polypeptide + H2)  Peptides
 Lyases. catalyze the addition or removal of groups (e.g., H2O, CO2, and NH3) to a double
bond.
Pyruvate formaldehyde + CO2
 Isomerases. Catalyze the inversion of asymmetric carbon atoms.
Exp : D –alanine  L -alanine
 Ligases. catalyze bond formation between two substrate molecules.
Pyruvate + HCO3  oxaloacetate
3. Discuss method to purify a protein.
1. Precipitation with high concentration of salt such as ammonium sulfate – to removes
the impurities
2. Dialysis – to remove salt
3. Chromatography
• Gel-filtration chromatography
• Ion-exchange chromatography
• Affinity chromatography
4. Electrophoresis – SDS gel electrophoresis
4. A scientist discovered a new seed storage protein, which they named BN, in the oilseed of
Brassica nigra. In order to isolate the protein, seeds were ground and extracted with water.
The proteins in the extract were precipitated with hydrochloric acid and the pellet isolated
by centrifugation was discarded. The supernatant was heated to 70°C to remove heat-labile
proteins, then lyophilized (freeze-dried). The lyophilized powder was dissolved in a small
amount of ammonium acetate buffer at pH = 5 and the sample was loaded onto a Sephadex
G-251 gel filtration column. The elution profile showed four peaks. Most of the BN protein
eluted in the first peak.
a. On what basis is separation achieved on the Sephadex gel filtration column?
Gel filtration chromatography separates proteins on the basis of molecular size,
which is generally proportional to molecular weight. In gel filtration
chromatography, the mobile phase consists of beads with pores that can
accommodate small molecules but not large molecules.
b. Compare the molecular size of BN protein with other three proteins found in the
Brassica nigra seeds.
The BN protein must be larger than the other proteins in the B. nigra seeds, since
it elutes from the column first. Large proteins elute before smaller proteins because
the only path from the column for a protein too large to enter the pores of the beads
is in between the beads, and this occurs more rapidly.
Following gel filtration chromatography, the BN protein was further purified by dialysis
using tubing with a 6000-8000 molecular weight cut-off. Analysis using SDS-PAGE (in
the absence of β-mercaptoethanol) showed a single band. The results are shown in Figure
1.
c. Why is the BN protein more pure after the dialysis step?
In the dialysis procedure, the protein is placed inside dialysis tubing. The pores in the
dialysis tubing allow proteins with molecular weights smaller than 6000-8000 daltons
to pass through into the dialysis buffer, while proteins larger than this, such as the JC,
are retained. Thus, the JC solution in the dialysis bag is rid of small molecular weight
contaminant proteins. The purity of the protein is indicated by a single band on the
SDS-PAGE gel.
d. Determine the molecular weight of the BN protein by constructing a standard curve
(plot molecular weight of the standards vs migration distance in the gel). Why was
the gel run in the absence of β -mercaptoethanol?
Use a ruler to measure the migration distances of the standards and of the JC
protein. The values are shown in the table below. Then construct a standard curve by
plotting molecular weight vs.distance migrated. The plot is shown below, and the
equation of the line is shown on the plot.
Molecular weigh, kD
36
29
24
20.1
14.2
12.5
JC protein
Distance migrated, cm
0.3
1.4
2.6
3.5
5.4
6.1
5.0
SDS-PAGE of BN: Standard curve