Download 462a Reading and Homework Assignment 3

462aH Homework Assignment 6
Homework Due 10/4/06
Homework (10 points)
(1) According to figure 10-6, p50 for hemoglobin in muscle tissue at pH 7.6 is ~ 22.5 torr and at
pH 7.2, p50 ~ 34 torr. Sketch the Hill plots expected for hemoglobin at these two pHs.
Assume, at pH 7.6, that the apparent R-state p50 = 1.5 torr, and the apparent T-state p50 = 150
B
torr.
3
2
B
1
0
-1
-2
-3
0.01
0.1
1
10
100
1000
(2) Oxygen binding by the hemocyanin of the shrimp Callianassa has been measured. Using
the following data, prepare a Hill plot and determine (a) p50, (b) n (the Hill coefficient), and (c)
the minimum number of binding sites on the protein molecule.
pO2 (kPa)
Y
0.15
0.003
1.03
0.019
1.43
0.035
4.23
0.084
9.59
0.190
13.40
0.329
16.44
0.487
18.23
0.557
22.24
0.673
27.09
0.734
34.96
0.794
43.60
0.834
60.26
0.875
98.22
0.913
Hill plot for hemocyanin
1.5
1
log theta/(1-theta)
0.5
0
-1
-0.5
-0.5
0
0.5
1
1.5
2
2.5
-1
-1.5
-2
-2.5
-3
log pO2
a) When Y = 0.5, log Y/(1-Y) = 0. Therefore p50 = 16.5 kPa.
b) The Hill coefficient is determined from the slope where the curve is steeply rising ≈ 2.8
(values between 2.5 -3.5 are acceptable)
c) The minimum number of binding sites is the integer greater than your Hill coefficient. If nH
< 3, the minimum number of binding sites is 3. If nH =3, or if nH < 4, the minimum number of
binding sites is 4. (Actually, this is an immense protein, with 24 total binding sites.)
(3) How would oxygen binding differ in a mutant hemoglobin in which beta chain His HC3 (His
146) was changed to valine, as compared with the wild type protein?
His HC3 forms an ion pair with an aspartate (Asp FG1) in the FG loop in the T state. In the R
state, this ion pair is broken. Substitution of His 146 with valine would not allow the ion pair to
form with Asp FG1. It is expected to have no effect on the ion pair that forms from the carboxyterminal COO- to Lys C5 (since the carboxyl group in present no matter which amino acid is
there.) See Figs 5-9 and 5-10. Removing the His-Asp ion pair would reduce the cooperativity,
and destabilize the T form.
Hemoglobin is also expected to show less of an effect of pH on the binding of oxygen,
because the protonation state of this histidine controls whether this salt bridge will form, and
hence affects the T to R transition.
(4) A biochemist is attempting to separate a DNA-binding protein (protein X) from other
proteins in a solution. Only three other proteins (A, B, and C) are present. The proteins have the
following properties:
protein A
protein B
protein C
protein X
pI (isoelectric point)
7.4
3.8
7.9
7.8
Size (Mr)
82,000
21,500
23,000
22,000
Bind to DNA?
yes
yes
no
yes
What type of protein separation techniques might the biochemist use to separate:
(a) protein X from protein A?
(b) protein X from protein B?
(c) protein X from protein C?
Briefly justify your answers.
General Principle: covering the basic concept of the three types of chromatography discussed
(i.e. Gel-Filtration, Ion-Exchange, and Affinity)
(a) Gel-Filtration Chromatography: due to size (Mr) difference.
(b) Ion-Exchange Chromatography: due to charge (pI) difference
(c) Affinity Chromatography: due to X binds DNA while the C does not
(5) Molecular weight analysis of a protein yields the following information:
Solvent
Dilute Buffer
Urea
Urea + -mercaptoethanol
Molecular weight
160,000
40,000
15,000 and 25,000
What can you deduce about the protein’s quaternary structure?
Urea is a denaturing agent and -mercaptoethanol can reduce disulfide bonds. Thus, there are 4
subunits of 40,000; Each subunit is made up of a two polypeptides that are 15,000 and 25,000
covalently linked by disulfide bonds.
(6) Deduce the amino acid sequence of a simple polypeptide from the following results. (The
amino acids in parentheses, separated by commas, represent amino acids compositions, not
sequences.)
(A) acid hydrolysis:
(Ala2, Arg, Lys2, Met, Phe, Ser2)
(B) Carboxypeptidase A:
(Ala)
(Ala, Arg, Lys2, Met, Phe, Ser2)
(C) Trypsin:
(Ala, Arg)
(Lys, Phe, Ser)
(Lys)
(Ala, Met, Ser)
(D) CNBr:
(Ala, Arg, Lys2, Met, Phe, Ser)
(Ala, Ser)
(E) Thermolysin:
(Ala, Arg, Ser)
(Ala, Lys2, Met, Phe, Ser)
Ala-Arg-Ser-Phe-Lys-Lys-Met-Ser-Ala